Escherichia coli

J Mol Biol, 1998 Oct 9, 282(5), 959 - 67
Transmembrane helix tilting and ligand-induced conformational changes in the lactose permease determined by site-directed chemical crosslinking in situ; Wu J et al.; The N-terminal six transmenbrane helices (N6) and the C-terminal six transmembrane helices (C6) of the lactose permease of Escherichia coli, each with a Cys residue, were co-expressed independently, and crosslinking was studied . Proximity of paired Cys residues in helices II (position 49, 52, 53, 56, 57, 60, 63 or 67) and VII (position 227, 230, 231, 234, 238, 241, 242 or 245) or XI (position 350, 353, 354, 357, 361 or 364) was examined by using two homobifunctional thiol-specific crosslinking agents of different lengths (6 or 10 A) . The results demonstrate that a Cys residue placed in the periplasmic half of helix II (position 49, 52, 53 or 57) crosslinks to Cys residues in the periplasmic half of helix VII (position 241, 242 or 245) . In contrast, no crosslinking is evident with paired-Cys residues in the cytoplasmic halves of helices II (position 60, 63 or 67) and VII (position 227, 230, 231, 234 or 238) . Remarkably, a Cys residue in the cytoplasmic half of helix II (position 60, 63 or 67) crosslinks with a Cys residue in the cytoplasmic half of helix XI (position 350, 353 or 354), while paired-Cys residues at positions in the periplasmic halves of the two helices do not crosslink . Therefore, helix II is tilted in such a manner that the periplasmic end is close to helix VII, and the cytoplasmic end is close to helix XI . Furthermore, ligand-binding alters the crosslinking efficiency of paired-Cys residues in helices II and VII or XI, indicating that both interfaces are conformationally active . The results are consistent with the conclusion that ligand-binding induces a scissors-like movement of helices II and VII that increases interhelical distance by 3 to 4 A at the periplasmic ends and decreases the distance by 3 to 4 A at the approximate middle of the two transmembrane helices .

Biochemistry, 1998 Sep 29, 37(39), 13902 - 9
Importance of central alpha-helices of human apolipoprotein A-I in the maturation of high-density lipoproteins; Frank PG et al.; We have studied the role of amphipathic alpha-helices in the ability of apoA-I to promote cholesterol efflux from human skin fibroblasts and activate lecithin:cholesterol acyltransferase (LCAT) . Three apoA-I mutants were designed, each by deletion of a pair of predicted adjacent central alpha-helices {Delta(100-143), Delta(122-165), Delta(144-186)}, and expressed in Escherichia coli . This strategy was used to minimize disruption of the predicted secondary structure of the resulting protein . These three central deletion mutants have been previously shown to be expressed as stable folded proteins but to exhibit altered phospholipid-binding properties . When recombined with phospholipids to form homogeneous LpA-I containing equivalent amounts of POPC and tested for their ability to promote diffusional cholesterol efflux from normal {3H}cholesterol-labeled fibroblasts, each mutant and the wild-type recombinant protein (Rec.-apoA-I) promoted cholesterol efflux with very similar rates at all the concentrations tested . These experiments showed that all LpA-I could acquire cellular cholesterol with similar affinity and binding capacity . However, when the cell-incubated LpA-I were incubated with purified LCAT, two mutants, Delta(122-165) and Delta(144-186), appeared incapable of activating the enzyme . To directly determine their ability to activate LCAT, each mutant and the control were recombined with equivalent amounts of cholesterol and phospholipid and incubated with the purified enzyme . The results show that whereas deletion of residues 100-143 has little effect on LCAT activation, deletion of residues 122-165 or 144-186 results in an inability of the mutants to promote cholesterol esterification . In conclusion, our results show that no specific sequence in the central domain of apoA-I is required for efficient diffusional cholesterol efflux from normal fibroblasts; however, residues 144-186 appear critical for optimum LCAT activation and cholesteryl ester accumulation . Since deletion of residues 144-186 also perturbs phospholipid association and prevents the formation of large LpA-I particles {Frank, P . G., Bergeron, J., Emmanuel, F., Lavigne, J . P., Sparks, D . L., Denefle, P., Rassart, E., and Marcel, Y . L . (1997) Biochemistry 36, 1798-1806}, the data show that this pair of alpha-helices plays an important role in the maturation of HDL . Sequence analysis of these apoA-I helices further identifies specific residues that appear essential to this activity.

Biochemistry, 1998 Sep 29, 37(39), 13893 - 901
D221 in thymidylate synthase controls conformation change, and thereby opening of the imidazolidine; Sage CR et al.; In thymidylate synthase (TS), the invariant residue Asp-221 provides the only side chain that hydrogen bonds to the pterin ring of the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate . All mutants of D221 except cysteine abolish activity . We have determined the crystal structures of two ternary complexes of the Escherichia coli mutant D221N . In a complex with dUMP and the antifolate 10-propargyl-5,8-dideazafolate (CB3717), dUMP is covalently bound to the active site cysteine, as usual . CB3717, which has no imidazolidine ring, is also bound in the usual productive orientation, but is less ordered than in wild-type complexes . The side chain of Asn-221 still hydrogen bonds to N3 of the quinazoline ring of CB3717, which must be in the enol form . In contrast, the structure of D221N with 5-fluoro-dUMP and 5,10-methylene-5,6,7, 8-tetrahydrofolate shows the cofactor bound in two partially occupied, nonproductive binding sites . In both binding modes, the cofactor has a closed imidazolidine ring and adopts the solution conformation of the unbound cofactor . In one of the binding sites, the pterin ring is turned around such that Asn-221 hydrogen bonds to the unprotonated N1 instead of the protonated N3 of the cofactor . This orientation blocks the conformational change required for forming covalent ternary complexes . Taken together, the two crystal structures suggest that the hydrogen bond between the side chain of Asp-221 and N3 of the cofactor is most critical during the early steps of cofactor binding, where it enforces the correct orientation of the pterin ring . Proper orientation of the cofactor appears to be a prerequisite for opening the imidazolidine ring prior to formation of the covalent steady-state intermediate in catalysis.

Biochemistry, 1998 Sep 29, 37(39), 13871 - 81
The oligomycin sensitivity conferring protein of rat liver mitochondrial ATP synthase: arginine 94 is important for the binding of OSCP to F1; Golden TR et al.; The oligomycin sensitivity conferring protein (OSCP) is an essential subunit of the mitochondrial ATP synthase (F0F1) long regarded as being directly involved in the energetic coupling of proton transport to ATP synthesis . To gain insight into the function of OSCP, mutations were made in a highly conserved central region of the subunit, and the recombinant proteins were studied using several biochemical assays . Rat liver OSCP was expressed to high levels in Escherichia coli, solubilized from inclusion bodies, renatured, and purified to homogeneity . The recombinant protein was able to reconstitute oligomycin-sensitive ATPase activity to inner membrane vesicles depleted of F1 and OSCP, and bound to F1 with a stoichiometry of 1:1 . A novel fluorescence anisotropy assay was developed to study the affinity of binding of F1 to OSCP, providing a Kd value of 51 +/- 11 nM . Two highly conserved, charged residues (E91 and R94) which lie within the central region of OSCP were mutated, and the recombinant proteins (E91Q, R94Q, and R94A) were purified to homogeneity and judged by CD spectroscopy to have structures similar to that of the wild-type protein . Both R94 mutants demonstrated little or no binding to F1, while the E91Q bound in a manner identical to that of wild-type OSCP . Significantly, all three mutant proteins were able to reconstitute F1 with membranes and to confer oligomycin sensitivity to the same extent as wild-type OSCP . These results demonstrate that a single tight binding site exists on isolated rat liver F1 for OSCP, and implicate arginine 94 as playing a critical role in this site . In addition, these results indicate that this tight binding site is not required for conferral of oligomycin sensitivity to the reconstituted F0F1 complex.

Biochemistry, 1998 Sep 29, 37(39), 13800 - 6
A conserved aspartate residue, Asp187, is important for Na+-dependent proline binding and transport by the Na+/proline transporter of Escherichia coli; Quick M et al.; Asp187 in the Na+/proline transporter of Escherichia coli (PutP) is conserved within the Na+/solute cotransporter family . Information on the role of this residue has been gained by amino acid substitution analysis . PutP with Glu, Asn, or Cys in place of Asp187 catalyzed Na+-coupled proline uptake at 75%, 25%, and 1.5%, respectively, of the Vmax of PutP-wild-type while the apparent Km for proline was only slightly altered . Importantly, acetylation or amidoacetylation of an engineered transporter containing a single Cys at position 187 stimulated proline uptake . Strikingly, PutP-D187C exhibited high-affinity proline binding even at very low Na+ concentrations (2 microM) while proline binding to PutP-wild-type, -D187E, and -D187N was strictly dependent on the Na+ concentration . The apparent independence of proline binding from the Na+ concentration can at least partially be attributed to an enhanced Na+ affinity of PutP-D187C . In addition, reaction of PutP containing a single Cys at position 187 with N-ethylmaleimide was inhibited by Na+ but not by Li+ or proline . The results indicate that electrostatic interactions of the amino acid side chain at position 187 in PutP with other parts of the transporter and/or the coupling ion are crucial for active proline transport . It is suggested that Asp187 is located close to the pathway of the coupling ion through the membrane and may be involved in the release of Na+ on the cytoplasmic side of the membrane.

Biochemistry, 1998 Sep 29, 37(39), 13736 - 43
Effects of secondary structure on DNA and RNA cleavage by diplatinum(II); Carter PJ et al.; The photochemistry of Pt2(pop)44- with nucleic acids has been studied using radiolabeled oligomers of DNA and RNA and high-resolution electrophoresis (pop is P2O5H22-) . Photolysis of Pt2(pop)44- with duplex DNA produces an even cleavage ladder and relatively little enhancement of cleavage upon treatment with piperidine . In contrast, the cleavage pattern is far less regular with single-stranded DNA, and there is a large enhancement in cleavage upon treatment with piperidine . Accordingly, photolysis of Pt2(pop)44- with the DNA hairpin 5'-d{ATCCTATTTATAGGAT} produces a much larger piperidine enhancement at the loop and end nucleotides than in the stem . There is an additional piperidine enhancement that occurs selectively at guanine residues either in RNA or in DNA at low Mg2+ concentrations that is attributed to outer-sphere electron transfer on the basis of the known excited-state redox potentials of Pt2(pop)44- and the expected oxidative chemistry of guanine . The extent of guanine oxidation is higher compared to the extent of sugar oxidation at low Mg2+ concentrations, which can be attributed to a shallower distance dependence for electron transfer compared to that for atom transfer . The effects of Mg2+ and piperidine or aniline treatment were examined on stem-loop structures of DNA and RNA and gave partial images of the expected secondary structures.

Biochemistry, 1998 Sep 29, 37(39), 13710 - 9
The human immunodeficiency virus type 1 Vpu protein enhances membrane permeability; Gonzalez ME et al.; Infection of T lymphocytes by the human immunodeficiency virus causes drastic alterations in the intracellular cation content of the infected cells . The human immunodeficiency virus type 1 genome encodes several accessory proteins, including Vpu, an integral membrane protein that forms ion channels in planar lipid bilayers . The effect of Vpu on the permeability of the plasma membrane to several molecules has been analyzed . Expression of vpu in Escherichia coli cells increases membrane permeability to a number of molecules such as 2-nitrophenyl beta-D-galactopyranoside, uridine, the impermeable translation inhibitor hygromycin B, and lysozyme . In addition, transient expression of Vpu in eukaryotic COS cells enhances entry of charged molecules such as hygromycin B and neurobiotin into these cells . The effect of Vpu on cell membrane permeability resembles that reported for other membrane-active proteins from different animal viruses, including influenza M2, Semliki Forest virus 6K, and poliovirus 2B and 3A proteins.

Biochemistry, 1998 Sep 29, 37(39), 13604 - 13
Lys75 of Anabaena ferredoxin-NADP+ reductase is a critical residue for binding ferredoxin and flavodoxin during electron transfer; Martinez-Julvez M et al.; Previous studies, and the three-dimensional structure of Anabaena PCC 7119 ferredoxin-NADP+ reductase (FNR), indicate that the positive charge of Lys75 might be directly involved in the interaction between FNR and its protein partners, ferredoxin (Fd) and flavodoxin (Fld) . To assess this possibility, this residue has been replaced by another positively charged residue, Arg, by two uncharged residues, Gln and Ser, and by a negatively charged residue, Glu . UV-vis absorption, fluorescence, and CD spectroscopies of these FNR mutants (Lys75Arg, Lys75Gln, Lys75Ser, and Lys75Glu) indicate that all the mutated proteins folded properly and that significant protein structural rearrangements did not occur . Steady-state kinetic parameters for these FNR mutants, utilizing the diaphorase activity with DCPIP, indicate that Lys75 is not a critical residue for complex formation and electron transfer (ET) between FNR and NADP+ or NADPH . However, steady-state kinetic activities requiring complex formation and ET between FNR and Fd or Fld were appreciably affected when the positive charge at position of Lys75 was removed, and the ET reaction was not even measurable if a negatively charged residue was placed at this position . These kinetic parameters also suggest that it is complex formation that is affected by mutation . Consistent with this, when dissociation constants (Kd) for FNRox-Fdox (differential spectroscopy) and FNRox-Fdrd (laser flash photolysis) were measured, it was found that neutralization of the positive charge at position 75 increased the Kd values by 50-100-fold, and that no complex formation could be detected upon introduction of a negative charge at this position . Fast transient kinetic studies also corroborated the fact that removal of the positive charge at position 75 of FNR appreciably affects the complex formation process with its protein partners but indicates that ET is still achieved in all the reactions . This study thus clearly establishes the requirement of a positive charge at position Lys75 for complex formation during ET between FNR and its physiological protein partners . The results also suggest that the interaction of this residue with its protein partners is not structurally specific, since Lys75 can still be efficiently substituted by an arginine, but is definitely charge specific.

Biochemistry, 1998 Sep 29, 37(39), 13499 - 506
The active-site arginine of S-adenosylmethionine synthetase orients the reaction intermediate; Reczkowski RS et al.; S-Adenosylmethionine (AdoMet) synthetase catalyzes the formation of AdoMet and tripolyphosphate (PPPi) from ATP and L-methionine and the subsequent hydrolysis of the PPPi to PPi and Pi before product release . Little is known about the roles of active-site residues involved in catalysis of the two sequential reactions that occur at opposite ends of the polyphosphate chain . Crystallographic studies of Escherichia coli AdoMet synthetase showed that arginine-244 is the only arginine near the polyphosphate-binding site . Arginine-244 is embedded as the seventh residue in the conserved sequence DxGxTxxKxI which is also found at the active site of inorganic pyrophosphatases, suggesting a potential pyrophosphate-binding motif . Chemical modification of AdoMet synthetase by the arginine-specific reagents phenylglyoxal or p-hydroxyphenylglyoxal inactivates the enzyme . ATP and PPPi protect the enzyme from inactivation, consistent with the presence of an important arginine residue in the vicinity of the polyphosphate-binding site . Site-specific mutagenesis has been used to change the conserved arginine-244 to either leucine (R244L) or histidine (R244H) . In the overall reaction, the R244L mutant has the kcat reduced approximately 10(3)-fold, with a 7 to 10-fold increase in substrate Km values; the R244H mutant has an approximately 10(5)-fold decrease in kcat . In contrast, the kcat values for hydrolysis of added PPPi by the R244L and R244H mutants have been reduced by less than 2 orders of magnitude . In contrast to the wild-type enzyme in which 98% of the Pi formed originates as the gamma-phosphoryl group of ATP, in the R244L mutant the orientation of the PPPi intermediate equilibrates at the active site yielding equal amounts of Pi from the alpha- and gamma-phosphoryl groups of ATP . Thus, the active-site arginine has a profound role in the cleavage of PPPi from ATP during AdoMet formation and in maintaining the orientation of PPPi in the active site, while playing a lesser role in the subsequent PPPi hydrolytic reaction.

Transplantation, 1998 Sep 15, 66(5), 567 - 72
Alterations in mRNA for inducible and endothelial nitric oxide synthase and plasma nitric oxide with rejection and/or infection of allotransplanted lungs; Wang X et al.; BACKGROUND: Experiments were designed to determine expression of type II (iNOS) and type III (ecNOS) nitric oxide synthase in lung parenchyma and systemic endothelial cells with rejection and/or infection of single lung allografts . METHODS: After single lung allotransplantation, dogs were maintained on standard triple immunosuppressive therapy for 5 days and then placed into one of three groups . Group I (n=4) was maintained on immunosuppressants, group II (n=7) immunosuppression was withdrawn to allow acute rejection of the allograft, and group III (n=6) infection was induced by bronchoscopic inoculation of Escherichia coli . RESULTS: At postoperative days 7-9, no histological evidence of rejection or infection was observed in transplanted lungs of group I . In lungs of group II, rejection ranged from mild to severe; in lungs of group III, infection was severe . Some animals had both rejection and infection (n=8) and were studied separately . Plasma levels of nitric oxide increased comparably with rejection and/or infection compared to preoperative values . Expression of mRNA for ecNOS decreased significantly in lung parenchyma but not in aortic endothelial cells from dogs of groups II and III . However, expression of mRNA for iNOS increased with both rejection and/or infection in both lung parenchyma and aortic endothelial cells . CONCLUSIONS: iNOS is induced locally within the graft and systemically in aortic endothelial cells with rejection and/or infection of lung allografts . Plasma levels of nitric oxide are elevated with both rejection and infection and may not be useful in the differential diagnosis of these processes after lung transplantation.

Cell, 1998 Sep 18, 94(6), 829 - 39
Structure of type IIbeta phosphatidylinositol phosphate kinase: a protein kinase fold flattened for interfacial phosphorylation; Rao VD et al.; Phosphoinositide kinases play central roles in signal transduction by phosphorylating the inositol ring at specific positions . The structure of one such enzyme, type IIbeta phosphatidylinositol phosphate kinase, reveals a protein kinase ATP-binding core and demonstrates that all phosphoinositide kinases belong to one superfamily . The enzyme is a disc-shaped homodimer with a 33 x 48 A basic flat face that suggests an electrostatic mechanism for plasma membrane targeting . Conserved basic residues form a putative phosphatidylinositol phosphate specificity site . The substrate-binding site is open on one side, consistent with dual specificity for phosphatidylinositol 3- and 5-phosphates . A modeled complex with membrane-bound substrate and ATP shows how a phosphoinositide kinase can phosphorylate its substrate in situ at the membrane interface.

Dig Dis Sci, 1998 Sep, 43(9 Suppl), 160S - 166S
Effects of rebamipide on production of several cytokines by human peripheral blood mononuclear cells; Aihara M et al.; Recently, the relative contributions of local T helper cell responses of the Th1-type and Th2-type to the pathogenesis of gastritis and peptic ulcers associated with Helicobacter pylori infection have been examined . However, the results were controversial with respect to whether cellular immunity (Th1-type) or humoral immunity (Th2-type) responses predominate in H . pylori infection and with respect to how these responses may contribute to disease pathogenesis . In this study, we investigated the characteristics of the production of various cytokines induced by H . pylori or lipopolysaccharide (LPS), which was derived from H . pylori or Escherichia coli, in human peripheral blood mononuclear cells (PBMC) . Live H . pylori induced production of many cytokines, such as IL-1beta, IL-10, IL-8, IFN-gamma, and TNF-alpha, whereas we could not detect IL-2 or IL-4 . Moreover, we evaluated the effect of rebamipide on the production of several cytokines from PBMC induced by various stimuli . Rebamipide suppressed the production of IL-8, IL-10, TNF-alpha, and IL-1beta induced by H . pylori in a dose-dependent manner . On the other hand, the production of IL-12 induced by H . pylori showed a tendency to increase as a result of treatment of the cells with rebamipide . These results suggested that rebamipide might be effective in regulating cytokine responses in the H . pylori-infected host and maintaining host immunity . Moreover, it might contribute positively to disease progression and bacterial eradication.

Dig Dis Sci, 1998 Sep, 43(9 Suppl), 154S - 159S
Effect of rebamipide on liver damage and increased tumor necrosis factor in a rat model of endotoxin shock; Hong KW et al.; We investigated the effect of rebamipide, a novel antiinflammatory agent, on liver damage in a rat model of circulatory shock induced by bacterial endotoxin (E . coli lipopolysaccharide, LPS) . Endotoxemia for 6 hr resulted in a 5.9-fold rise in the serum levels of nitrite (P < 0.05) with a significant rise in the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactic dehydrogenase (LDH), suggestive of liver dysfunction . The increased activities of serum ALT, AST, and LDH, but not serum nitrite were significantly inhibited by rebamipide (100 mg/kg, orally for five days) . Myeloperoxidase activity in the liver was significantly elevated in the rats with endotoxemia by 2.4-fold (P < 0.05), which was also significantly inhibited by rebamipide . Upon LPS injection, serum TNF-alpha levels peaked at 1 hr after LPS (from 167.4 +/- 20.0 to 1570.0 +/- 100.0 pg/ml) and thereafter rapidly declined . The increased TNF-alpha level measured at 1 hr was significantly inhibited by pretreatment with rebamipide (100 mg/kg for five days) . It is suggested that rebamipide exerts a strong protective effect on the LPS-induced liver damage through inhibition of activation of neutrophils and TNF-alpha production.

Eur J Neurosci, 1998 Mar, 10(3), 989 - 99
Regulation of cell-type specific expression of lacZ by the 5'-flanking region of mouse GAD67 gene in the central nervous system of transgenic mice; Katarova Z et al.; The transcriptional regulation of the murine gene encoding the 67-kDa form of glutamic acid decarboxylase (GAD67) was studied by beta-galactosidase histochemistry in transgenic mice carrying fusion genes between progressively longer portions of the 5'-upstream regulatory region of GAD67 and E . coli lacZ . No expression was detected in brains of mice carrying 1.3 kb of upstream sequences including a housekeeping and two conventional promoters, and two negative regulatory elements with homology to known silencers . In mice carrying the same portion of the promoter region plus the first intron, lacZ expression in the adult central nervous system was found in few, exclusively neuronal sites . The number of correctly stained GABAergic centres increased dramatically with increasing the length of the 5'-upstream region included in the construct which suggests that multiple putative spatial enhancers are located in this region . Their action is influenced by epigenetic mechanisms that may be due to site-of-integration and transgene copy-number effects . Additional cis-acting elements are needed to obtain fully correct expression in all GABAergic neurons of the adult central nervous system.

FEMS Immunol Med Microbiol, 1998 Aug, 21(4), 313 - 21
In vitro inhibition of adhesion of enterotoxigenic Escherichia coli K88 to piglet intestinal mucus by egg-yolk antibodies; Jin LZ et al.; The objective of the study was to determine if the adhesion of E . coli K88 to piglet intestinal mucus could be inhibited in vitro by spray-dried egg-yolk anti-K88 antibodies . Binding of E . coli was monitored using a radioactive assay . Four 14+/-2-day-old healthy piglets were used for the preparation of mucus from the small intestine . Competition and displacement phenomena were investigated by incubating (a) egg-yolk antibodies and E . coli together prior to adding to the mucus and (b) E . coli and mucus, followed by egg-yolk antibodies . The results demonstrated that egg-yolk antibodies inhibited the adhesion of 3H-labeled local strain of hemolytic E . coli K88+ (E . coli K88+ MB) to piglet small intestinal mucus by 84.6-97.0% when the egg-yolk antibodies were diluted 10, 20, 40 or 100 times . The adhesion inhibiting effects of egg-yolk antibodies declined dramatically when the antibody dilution was more than 250-fold . A similar adhesion inhibiting effect was observed when egg-yolk antibodies were incubated with E . coli K88+ MB for 15, 30 and 60 min prior to the adhesion test . Egg-yolk antibodies when diluted 50- and 100-fold had a very strong inhibiting ability against E . coli K88+ MB at a concentration of 10(9) colony forming units (cfu) ml(-1) (adhesion was < 6%) . However, dilution of 100 times for egg yolk antibodies was insufficient to inhibit the adhesion of E . coli K88+ MB to intestinal mucus when the concentration of E . coli K88+ MB was 10(10) cfu ml(-1) . The displacement test indicated that there was no significant reduction in the adhesion of E . coli K88+ MB to the small intestinal mucus when egg-yolk antibodies were added after adhesion of the organism to the mucus . These studies demonstrate that anti-K88+ MB fimbriae antibodies from chicken egg-yolk when added to E . coli K88+ MB prevented their binding to receptors in the mucus isolated from the intestine of piglets.

Chirurgie, 1998 Apr, 123(2), 168 - 74
{Gene therapy of cerebral glioblastoma by adenovirus vector . Experimental model in the rat}; Dufour T et al.; Our aim was to test the therapeutic effects of adenovirus-mediated gene therapy in an animal brain tumor model which was obtained by stereotactic injection of 9L gliosarcoma cells into the caudate nucleus of rat brains . Seven days after the implantation of tumor cells, adenovirus vectors bearing the Escherichia coli beta-galactosidase gene (ADVbgal) or the herpes simplex virus thymidine kinase gene (ADVtk) were stereotactically injected into the tumor . Injection of the ADVbgal resulted in the expression of the marker gene in 11 animals . Transfer of the ADVtk was followed, 3 days later, by intraperitoneal injection of ganciclovir (GCV) for 10 days . A control group was treated with saline instead of GCV . We observed a significant regression of the tumors in the rats treated with ADVtk and GCV as compared with control animals . In four cases the tumor completely disappeared after treatment . These results demonstrate the potential efficacy of adenovirus-mediated transfer of the HSVtk gene following by GCV administration for the treatment of glioblastomas.

J Biomol NMR, 1998 Aug, 12(2), 319 - 24
Rapid measurement of scalar three-bond 1HN-1H alpha spin coupling constants in 15N-labelled proteins; Ponstingl H et al.; Two new 2D NMR experiments, CT-HMQC-HA and CT-HMQC-HN, are proposed for the rapid measurement of homonuclear 3JHNH alpha coupling constants of uniformly 15N-enriched proteins in solution . The experiments are based on the comparison of the signal intensities in a pair of constant-time {15N,1H}-HMQC spectra recorded with and without decoupling of the amide proton-alpha proton coupling . Experimental data recorded with the 78-residue N-terminal domain of the E . coli arginine repressor (ArgR-N) and with oxidized E . coli flavodoxin (176 residues) showed good agreement with 3JHNH alpha coupling constants obtained by fitting of the multiplet fine structure of the amide proton resonances or from a 3D HNHA-J experiment, respectively . Quantitative estimates for the effects from different relaxation rates of in-phase and antiphase magnetization are given.

J Biomol NMR, 1998 Aug, 12(2), 299 - 306
An asymmetric deuterium labeling strategy to identify interprotomer and intraprotomer NOEs in oligomeric proteins; Jasanoff A; A major difficulty in determining the structure of an oligomeric protein by NMR is the problem of distinguishing inter- from intraprotomer NOEs . In order to address this issue in studies of the 27 kD compact trimeric domain of the MHC class II-associated invariant chain, we compared the 13C NOESY-HSQC spectrum of a uniformly 13C-labeled trimer with the spectrum of the same trimer labeled with 13C in only one protomer, and with deuterium in the other two protomers . The spectrum of the unmixed trimer included both inter- and intraprotomer NOEs while the spectrum of the mixed trimer included only intraprotomer peaks . NOEs clearly absent from the spectrum of the mixed trimer could be confidently assigned to interprotomer interactions . Asymmetrically labeled trimers were isolated by refolding a 13C-labeled shorter form of the protein with a 2H-labeled longer form, chromatographically purifying trimers with only one short chain, and then processing the trypsin to yield only protomers with the desired N- and C-termini . In contrast to earlier studies, in which statistical mixtures of differently labeled protomers were analyzed, our procedure generated only a well-defined 1:2 oligomer, and no other mixed oligomers were present . This increased the maximum possible concentration of NMR-active protomers and thus the sensitivity of the experiments . Related methods should be applicable to many oligomeric proteins, particularly those with slow protomer exchange rates.

J Biomol NMR, 1998 Aug, 12(2), 259 - 76
Some NMR experiments and a structure determination employing a {15N,2H} enriched protein; Mal TK et al.; We present the results of studies of an aqueous sample of a highly {15N,2H} enriched protein, the SH3 domain from Fyn . Measurements of 1H relaxation and interactions between H2O solvent and exchangeable protons are given, as well as a method for increasing the effective longitudinal relaxation of solvent exchangeable proton resonances . The long-range isotope shifts are measured, for 1H and 15N, which arise due to perdeuteration . Simulations, which employed a 7 or 8 spin relaxation matrix analysis, were compared to the experimental data from a time series of 2D NOESY datasets for some resonances . The agreement between experiment and simulation suggest that, with this 1H dilute sample, relatively long mixing times (up to 1.2 s) can be used to detect specific dipolar interactions between amide protons up to about 7A apart . A set of 155 inter-amide NOEs and 7 side chain NOEs were thus identified in a series of 3D HSQC-NOESY-HSQC experiments . These data, alone and in combination with previously collected restraints, were used to calculate sets of structures using X-PLOR . These results are compared to the available X-ray and NMR structures of the Fyn SH3 domain.

J Biomol NMR, 1998 Aug, 12(2), 209 - 22
Hydrogen bonding geometry of a protein-bound carbohydrate from water exchange-mediated cross-relaxation; Sayers EW et al.; We present heteronuclear two-dimensional methods for the analysis of the geometry of exchangeable protons on a protein-bound carbohydrate . By using a water-selective NOESY-HSQC, we observed cross-relaxation between carbohydrate hydroxyl protons and non-exchangeable ring protons in the complex of {13C6}-alpha-methyl-D-mannopyranoside with recombinant rat mannose binding protein . Using a simple kinetic model, we were able to explain the differences in the initial slopes of the resulting cross-relaxation buildup curves in terms of the geometry of the hydroxyl protons in the bound state . The hydroxyl rotamers consistent with our cross-relaxation data fit very well with predictions based on the crystal structure of MBP bound to a mannose-rich oligosaccharide . These methods should be applicable to other systems where both ligand exchange and water exchange are fast relative to the rate of cross-relaxation.

Gene, 1998 Sep 18, 218(1-2), 57 - 61
A new set of positive/negative selectable markers for mammalian cells; Karreman C; Five new positive and negative selectable markers were created for use in mammalian cells . Their negative selectabilities are based on the Thymidine kinase (Tk) gene of Herpes Simplex virus (HSV) or the Cytidine deaminase (codA) gene of E . coli . The markers can be selected positively by their ability to induce either Hygromycin (Hyg), neomycin (neo), puromycin (PAC) or Blasticidin S (BlaS) resistance . With these markers, two complete sets of markergenes are available that induce independent negative selectable phenotypes.

Gene, 1998 Sep 18, 218(1-2), 49 - 56
Sequence and activity of parathyroid hormone/parathyroid hormone-related protein receptor promoter region in human osteoblast-like cells; Manen D et al.; The parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor gene has been characterized in various species . The structure of its promoter and the regulation of its expression in human tissues have, however, not been clearly established yet . We characterized the region upstream of the PTH/PTHrP receptor gene and investigated its promoter activity in the human osteoblast-like SaOS-2 cells . In this region, three untranslated exons were localized, U1 and U2 by using a kidney cDNA, and U3 by homology with the mouse gene . In human osteoblast-like cells, a distal promoter (P1) was found to be inactive, as evaluated by luciferase reporter gene assays, in contrast to the situation found in human and mouse kidney tissue . A second promoter (P2), previously described in mouse and human kidney, was shown to be active in human osteoblast-like SaOS-2 cells . We found a hitherto uncharacterized promoter (P3), closely upstream of the ATG start codon . The activities of P2 and P3 were not additive . These results provide important information on the structure of the 5' flanking region of the human PTH/PTHrP gene receptor.

Proc Natl Acad Sci U S A, 1998 Sep 29, 95(20), 11897 - 902
Characterization of hematopoietic progenitor cells that express the transcription factor SCL, using a lacZ "knock-in" strategy; Elefanty AG et al.; Gene targeting experiments have demonstrated that the transcription factor SCL is essential for primitive and definitive hematopoiesis in the mouse . To study the functional properties of hematopoietic cells expressing SCL, we have generated mutant mice (SCLlacZ/w) in which the Escherichia coli lacZ reporter gene has been "knocked in" to the SCL locus, thereby linking beta-galactosidase expression to transcription from the SCL promoter . Bone marrow cells from heterozygous SCLlacZ/w mice were sorted into fractions expressing high, intermediate and low levels of beta-galactosidase (designated lacZhigh, lacZint, and lacZneg) . Cells that were lacZhigh or lacZint were enriched for day 12 spleen colony-forming units and myeloid and erythroid colony-forming cells (CFCs) . These fractions included >99% of the erythroid and >90% of the myeloid CFCs . Culture of sorted bone marrow populations on stromal cells secreting interleukin-7 or in fetal thymic organ cultures showed that B and T lymphoid progenitors were also present in the lacZhigh and lacZint fractions . These data provide a functional correlation between SCL expression and colony-forming ability in immature hematopoietic cells . Our data also suggested that expression of SCL was transient and confined to hematopoietic stem and/or progenitor cells, because the differentiated progeny of most lineages (except the erythroid) were beta-galactosidase-negative.

Proc Natl Acad Sci U S A, 1998 Sep 29, 95(20), 11643 - 8
Thermodynamics of electron transfer in Escherichia coli cytochrome bo3; Schultz BE et al.; The proton translocation mechanism of the Escherichia coli cytochrome bo3 complex is intimately tied to the electron transfers within the enzyme . Herein we evaluate two models of proton translocation in this enzyme, a cytochrome c oxidase-type ion-pump and a Q-cycle mechanism, on the basis of the thermodynamics of electron transfer . We conclude that from a thermodynamic standpoint, a Q-cycle is the more favorable mechanism for proton translocation and is likely occurring in the enzyme.

Nature, 1998 Sep 17, 395(6699), 244 - 50
Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d; Brotherton DH et al.; The crystal structure of the cyclin D-dependent kinase Cdk6 bound to the p19 INK4d protein has been determined at 1.9 A resolution . The results provide the first structural information for a cyclin D-dependent protein kinase and show how the INK4 family of CDK inhibitors bind . The structure indicates that the conformational changes induced by p19INK4d inhibit both productive binding of ATP and the cyclin-induced rearrangement of the kinase from an inactive to an active conformation . The structure also shows how binding of an INK4 inhibitor would prevent binding of p27Kip1, resulting in its redistribution to other CDKs . Identification of the critical residues involved in the interaction explains how mutations in Cdk4 and p16INK4a result in loss of kinase inhibition and cancer.

Nature, 1998 Sep 17, 395(6699), 237 - 43
Structural basis for inhibition of the cyclin-dependent kinase Cdk6 by the tumour suppressor p16INK4a; Russo AA et al.; The cyclin-dependent kinases 4 and 6 (Cdk4/6) that control the G1 phase of the cell cycle and their inhibitor, the p16INK4a tumour suppressor, have a central role in cell proliferation and in tumorigenesis . The structures of Cdk6 bound to p16INK4a and to the related p19INK4d reveal that the INK4 inhibitors bind next to the ATP-binding site of the catalytic cleft, opposite where the activating cyclin subunit binds . They prevent cyclin binding indirectly by causing structural changes that propagate to the cyclin-binding site . The INK4 inhibitors also distort the kinase catalytic cleft and interfere with ATP binding, which explains how they can inhibit the preassembled Cdk4/6-cyclin D complexes as well . Tumour-derived mutations in INK4a and Cdk4 map to interface contacts, solidifying the role of CDK binding and inhibition in the tumour suppressor activity of p16INK4a.

Cancer Lett, 1998 Aug 14, 130(1-2), 127 - 31
Stannous chloride and the glucoheptonic acid effect: study of a kit used in nuclear medicine; Assis ML et al.; Stannous dichloride is used as a reducing agent in the preparation of technetium-99m radiopharmaceuticals . We decided to evaluate the genotoxic potential of the tin (II)-glucoheptonate complex in the kit using a tester strain of Escherichia coli AB1157 . Our results show that tin (II) chloride and the tin (II)-glucoheptonate complex exert a genotoxic effect in this system . While the genotoxic effect disappeared when the glucoheptonate concentration was increased, the glucoheptonate did not protect the cultures from the damaging effects of hydrogen peroxide . The ability of glucoheptonate to protect cultures from tin (II)-induced damage can be explained on the basis of its metal chelating properties.

J Neurochem, 1998 Oct, 71(4), 1369 - 80
Cloning and characterization of a novel form of tyrosine hydroxylase from the human parasite, Schistosoma mansoni; Hamdan FF et al.; Catecholamines such as dopamine and noradrenaline play important roles as neuromuscular transmitters and modulators in all parasitic helminths, including the human parasite, Schistosoma mansoni . We have cloned a novel S . mansoni tyrosine hydroxylase (SmTH) cDNA that shows high homology to mammalian tyrosine hydroxylase, the enzyme that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines . Two subsets of SmTH transcripts were identified, one of which carries the S . mansoni spliced-leader (SL) sequence at its 5' end, whereas the other does not appear to be trans-spliced to the S . mansoni SL . The two types of SmTH transcripts encode the same protein of 465 amino acids and a predicted size of 54 kDa . Expression of SmTH as an N-terminal histidine fusion protein in Escherichia coli produced an active enzyme that was purified approximately 52-fold to apparent homogeneity and had a final specific activity of 0.78 micromol/min/mg . The purified enzyme was found to have the same absolute requirement for a tetrahydrobiopterin cofactor and the same sensitivity to inhibition by high concentrations of the substrate, tyrosine, as the mammalian enzyme . Purified SmTH also showed characteristic inhibition by catecholamine products, although the sensitivity to product inhibition was lower than that of the mammalian enzyme . This evidence indicates that SmTH encodes a functional tyrosine hydroxylase that has catalytic properties similar to those of the mammalian host's enzyme but may differ in its properties of regulation . This first demonstration of tyrosine hydroxylase in a parasitic helminth further suggests that the parasites have the enzymatic capacity to synthesize catecholamines endogenously.

Methods Enzymol, 1998, 295, 424 - 50
Energetic methods to study bifunctional biotin operon repressor; Beckett D; Application of a broad range of approaches and techniques to analysis of the functional energetics of the biotin regulatory system has enabled dissection of each of the steps in the assembly of this transcriptional repression complex . Although the molecular details of the interactions are not yet completely understood, the studies described in this article have laid a solid foundation for future studies of the system . The application of kinetic and equilibrium methods to studies of binding of the allosteric effector has allow determination of the kinetic parameters governing the interaction of the protein and ligand . The kinetic parameters have, furthermore, been utilized to calculate the equilibrium parameters associated with the binding . The great advantage of using kinetic methods to study the binding process is the additional information provide about the mechanism of allosteric activation of the protein . Based on the initial observation of a kinetic time course that is consistent with the occurrence of a structural change concomitant with effector binding, additional measurements have been performed that have allowed formulation of a testable hypothesis concerning the nature and location of one locus: the structural change in the three-dimensional structure of BirA . Studies of assembly of the protein indicate the bio-5-AMP is an allosteric activator of dimerization of the protein . The dimerization is, however, weak . These results have been critical in analyzing site-specific DNA binding measurements . Application of the DNase I footprinting technique has allowed formulation of a model for association of holoBirA with bioO . Results of studies of binding of the protein to mutant operator templates, although not yielding the anticipated results, provide further insight into the mechanism of association of the protein and DNA . Two models for binding, the validity of which can be tested via the application of kinetic techniques, have been derived from these measurements . The results of quantitative studies of the biotin regulatory system can be interpreted in the context of the biological function of the system . The biotin holoenzyme ligases are a class of enzymes found across the evolutionary spectrum . Only a subset of these enzymes, including BirA, also function as transcriptional repressors . The tight binding of the allosteric effector may be understood in light of the bifunctional nature of the BirA-bio-5'-AMP complex . It is possible that the unusually high thermodynamic and kinetic stability of the complex ensures that the most probable state of the protein in vivo is the adenylate-bound form . This complex, not the unliganded protein, is active in both enzymatic transfer of biotin and site-specific DNA binding . This ensures that on depletion of the intracellular pool of apoBCCP, BirA-bio-5'-AMP accumulates and binds to bioO to repress transcription of the biotin biosynthesis operon . The intracellular demand for and synthesis of biotin are, consequently, tightly coupled in the system . The dimerization that accompanies adenylate binding to BirA appears to be significant for site-specific binding of the protein to bioO . Functionally, the simultaneous binding of the two monomers to the two operator half-sites, regardless of the kinetic mechanism by which it occurs, ensures coordinate regulation of transcription initiation from both biotin operon promoters . The multifaceted approach utilized in studies of the biotin regulatory system can serve as a model for studies of any complex transcriptional regulatory system . It is critical in elucidating the functional energetics of any of these systems that the assembly first be dissected into the constituent interactions and that each of these interactions be studied in isolation . This is not only critical for understanding the physicochemical properties of each individual contributing interaction, but is also a necessary precursor to studies of thermodynamic linkage in the system . (AB

Arch Biochem Biophys, 1998 Oct 1, 358(1), 182 - 8
The N-terminal region is important for the allosteric activation and inhibition of the Escherichia coli ADP-glucose pyrophosphorylase; Wu MX et al.; The ADPglucose pyrophosphorylase (EC 2.7.7.27) from Escherichia coli is allosterically activated by fructose 1,6-bisphosphate and inhibited by AMP . Proteolysis of the enzyme with proteinase K causes loss of activity and generates two peptides, 21 and 28 kDa, from the 49.7-kDa subunit . The presence of ADPglucose, Mg2+, and fructose 1, 6-bisphosphate during the incubation with proteinase K protected the enzyme activity and prevented cleavage at sites Met181-Ala182 and Phe192-Val193 . Proteolysis of the protected enzyme removed 10 to 13 amino acids from the N-terminal and 2 amino acids from the C-terminal . The resulting enzyme was almost independent of the need for fructose 1,6-bisphosphate for maximal activity and insensitive to inhibition by AMP . The apparent affinity for the substrates was similar to that of the fully-activated wild-type enzyme . These data suggest that amino acid residues in the N-terminal portion and possibly the C-terminal portion of ADPglucose pyrophosphorylase are part of the regulatory domain of the enzyme, critical for allosteric regulation of the enzyme .

Arch Biochem Biophys, 1998 Oct 1, 358(1), 104 - 15
Engineering of pyridine nucleotide specificity of nitrate reductase: mutagenesis of recombinant cytochrome b reductase fragment of Neurospora crassa NADPH:Nitrate reductase; Shiraishi N et al.; The cytochrome b reductase fragment of Neurospora crassa NADPH:nitrate reductase (EC 1.6.6.3) was overexpressed in Escherichia coli with a His-tag for purification after mutation of the NADPH binding site . The recombinant enzyme fragment was altered by site-directed mutagenesis guided by the three-dimensional structure of cytochrome b reductase fragment of corn NADH:nitrate reductase (EC 1.6.6.1) . Substitution of Asp for Ser920 (using residue numbering for holo-NADPH:nitrate reductase of N . crassa) greatly increased preference for NADH . This mutant had nearly the same NADH:ferricyanide reductase kcat as wild-type with NADPH . Substitutions for Arg921 had little influence on coenzyme specificity, while substitution of Ser or Gln for Arg932 did . The cytochrome b reductase mutant with greatest preference for NADH over NADPH was the doubly substituted form, Asp for Ser920/Ser for Arg932, but it had low activity and low affinity for coenzymes, which indicated a general loss of specificity in the binding site . Steady-state kinetic constants were determined for wild type and mutants with NADPH and NADH . Wild type had a specificity ratio of 1100, which was defined as the catalytic efficiency (kcat/Km) for NADPH divided by catalytic efficiency for NADH, while Asp for Ser920 mutant had a ratio of 0.17 . Thus, the specificity ratio was reversed by over 6000-fold by a single mutation . Preference for NADPH versus NADH is strongly influenced by presence/absence of a negatively charged amino acid side chain in the binding site for the 2' phosphate of NADPH in nitrate reductase, which may partially account for existence of bispecific NAD(P)H:nitrate reductases (EC 1.6.6.2) .

Anal Biochem, 1998 Oct 1, 263(1), 51 - 6
A continuous fluorimetric assay for tail-specific protease; Beebe KD et al.; A continuous fluorimetric assay for tail-specific protease (Tsp) has been developed using a fluorescence donor/quencher system, in which 5-{(2-aminoethyl) amino}naphthalene-1-sulfonic acid (EDANS) and 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) are attached to the N-terminus and the lysyl side chain of peptide AARAAK-(6-aminocaproyl)2-ENYALAA, respectively . Tsp-mediated cleavage of the Ala-Arg peptide bond separates the quencher, DABCYL, from the donor, EDANS, and results in a large increase in the fluorescent yield of EDANS (>50-fold) . Using this sensitive assay, Escherichia coli tail-specific protease was shown to exhibit typical Michaelis-Menten kinetics with a kcat of 0.086 +/- 0.002 s-1, KM of 4.0 +/- 0.3 microM, and kcat/KM of 2.2 x 10(4) M-1 s-1 . A control substrate, which only differs from the above substrate by having a charged residue (glutamate) at the C-terminus, showed drastically reduced activity to Tsp (kcat/KM = 58 M-1 s-1) . A peptide containing the C-terminal sequence of the substrate, GRGYALAA, was shown to be a competitive inhibitor of Tsp with a KI value of 31 microM . These results demonstrate the utility of this assay for the rapid assessment of Tsp activity .

Anal Biochem, 1998 Sep 10, 262(2), 122 - 8
Site-specific, enzymatic biotinylation of recombinant proteins in Spodoptera frugiperda cells using biotin acceptor peptides; Duffy S et al.; Site-specific, enzymatic biotinylation of recombinant proteins can be exploited to circumvent many problems associated with the use of biotinylating reagents in vitro and to overcome some of their inherent limitations . Additionally, biotinyl proteins can be purified to near-homogeneity in a single step under native conditions . Here we report that a biotin acceptor peptide (BAP) substrate for Escherichia coli biotin holoenzyme synthetase (BirA) can be used to label recombinant proteins with biotin in Spodoptera frugiperda (Sf9) cells, and we describe a collection of baculovirus transfer vectors specifically designed for this purpose . These BioBac vectors will greatly expand the range of proteins to which this technology can be applied .

Anal Biochem, 1998 Sep 10, 262(2), 110 - 21
Identification of biotinylation sites on proteins by selective retrieval of 2-iminobiotinylated peptides from proteolytic peptide mixtures: localization of the accessible lysine residues on the photosystem I subunits PsaD and PsaE; von Leoprechting A et al.; An affinity purification technique was established that allows the selective isolation of 2-iminobiotinylated peptides from proteolytic digest of proteins in order to identify surface-exposed protein domains . Serving as model systems, two photosystem I subunits, PsaD and PsaE from the cyanobacterium Synechococcus elongatus, were overexpressed in Escherichia coli, modified in vitrowith NHS-2-iminobiotin which incorporates 2-iminobiotin at exposed amino groups, and subjected to proteolytic digestion by Glu-C and Arg-C protease, respectively . 2-Iminobiotin-containing proteolytic peptides were subsequently extracted from the proteolytic digests using avidin agarose in a batch procedure and the extracted peptides were separated by HPLC chromatography . The analysis of the peptide maps by electrospray ionization mass spectrometry or N-terminal sequencing showed that avidin-extracted peptide fractions contain almost exclusively 2-iminobiotinylated proteolytic fragments of PsaE or PsaD . No unmodified peptides of PsaD or PsaE were detected . According to this analysis, PsaE is accessible to biotinylation at all of its 7 lysine residues and at its N-terminus . Similarly, all 11 lysine residues of PsaD can be biotinylated and only the N-terminus of PsaD is not accessible .

Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7441 - 5
Photorepair mutants of Arabidopsis; Jiang CZ et al.; UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6-4) pyrimidinone dimer (6-4 product) . Although Escherichia coli and Saccharomyes cerevisiae produce a CPD-specific photolyase that eliminates only this class of dimer, Arabidopsis thaliana, Drosphila melanogaster, Crotalus atrox, and Xenopus laevis have recently been shown to photoreactivate both CPDs and 6-4 products . We describe the isolation and characterization of two new classes of mutants of Arabidopsis, termed uvr2 and uvr3, that are defective in the photoreactivation of CPDs and 6-4 products, respectively . We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase . In addition, we are able to generate plants in which only CPDs or 6-4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers . This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system.

Protein Eng, 1998 Aug, 11(8), 707 - 13
Effects of the length of a glycine linker connecting the N-and C-termini of a circularly permuted dihydrofolate reductase; Iwakura M et al.; An important consideration in the construction of active and stable circularly permuted proteins is the connective sequence that links the native N- and C-termini . For this reason, various lengths of polyglycine linkers (two, three, four, five and six glycines) were employed to connect the original N- and C-termini of a circularly permuted construct of Escherichia coli dihydrofolate reductase (DHFR) in which the new N-terminus was Met16 . Examination of the circular dichroism (CD) spectra, gel-filtration chromatography elution profiles, urea-induced unfolding properties and enzyme kinetics revealed that, among the linkers tested, a linker length of five glycines was the most favorable . The Vmax of the circularly permuted variant with a five glycine linker (cpM16G5) was about 20% that of wild-type DHFR, although far UV CD spectra, gel filtration elution time, conformational stability and Km for the substrate dihydrofolate and Kd for the coenzyme NADPH were comparable in the two proteins . Another circularly permuted DHFR with a five glycine linker in which a new N-terminus was created at Leu24 (cpL24G5) was also constructed and assayed . The Vmax of cpL24G5 was almost the same as the wild-type, presumably due to the optimization of the glycine linker . The improved activity of the Leu24 permutant is probably due to the disruption of a catalytically important structure, the M20 loop, in the Met16 permutant.

Protein Eng, 1998 Aug, 11(8), 683 - 90
Calorimetric study of mutant human lysozymes with partially introduced Ca2+ binding sites and its efficient refolding system from inclusion bodies; Koshiba T et al.; During the process of evolution, ancestral lysozymes evolved into calcium-binding lysozymes by acquiring three critical aspartate residues at positions 86, 91 and 92 . To investigate the process of the acquisition of calcium-binding ability, two of the aspartates were partially introduced into human lysozyme at positions 86, 91 and 92 . These mutants (HLQ86D, HLA92D and HLQ86D/D91Q/A92D), having two critical aspartates in calcium-binding sites, were expressed in Escherichia coli as non-active inclusion bodies . For the preparation of lysozyme samples, a refolding system using thioredoxin was established . This system allowed for effective refolding of wild-type and mutant lysozymes, and 100% of activity was recovered within 4 days . The calcium ion dependence of the melting temperature (Tm) of wild-type and mutant lysozymes was investigated by differential scanning calorimetry at pH 4.5 . The Tm values of wild-type, HLQ86D and HLA92D mutants were not dependent on calcium ion concentration . However, the Tm of HLQ86D/D91Q/A92D was 4 degrees higher in the presence of 50 mM CaCl2 than in its absence, and the calcium-binding constant of this mutant was estimated to be 2.25(+/-0.25)x10(2) M(-1) at pH 4.5 . Moreover, the calcium-binding ability of this mutant was confirmed by the result using Sephadex G-25 gel chromatography . These results indicate that it is indispensable to have at least two aspartates at positions 86 and 92 for acquisition of calcium-binding ability . The process of the acquisition of calcium-binding site during evolution of calcium-binding lysozyme is discussed.

Protein Eng, 1998 Aug, 11(8), 627 - 30
Misleading local sequence alignments: implications for comparative protein modelling; Saqi MA et al.; Although it is well known that significant sequence similarity between proteins is reflected at the structural level, it is commonly assumed that any misaligned regions, as judged by the correct structure based alignment, are those where the local sequence identity is lower than the global . Recent studies have shown that this is not always the case and there can exist short stretches of high local identity which is not reflected in the structure based alignment . An analysis is presented of 290 pairs of homologous proteins with a view to quantifying the occurrence of these misleading local sequence alignments (MLSAs) . It is found that such MLSAs are likely if the global sequence identity is less than 40% and can occur even when it is greater than 60% . The results have implications for automated homology modelling and also for the inference of function made by comparison.

Mol Gen Genet, 1998 Aug, 259(3), 336 - 44
Orientation-dependent enhancement by H-NS of the activity of the type 1 fimbrial phase switch promoter in Escherichia coli; Schembri MA et al.; Phase variation of type 1 fimbriation in Escherichia coli is associated with the inversion of a 314-bp DNA element, positioned proximal to and upstream of fimA, which encodes the major type 1 fimbrial subunit . This DNA switch region contains a promoter that drives transcription of fimA only when the switch is in the ON orientation . Using chromosomal and plasmid-borne lacZ reporter cassettes, we show here how the global regulator H-NS affects the activity of the fimA promoter . In phase-locked reporter cassettes the activity of the fimA promoter was found to be enhanced in a hns-positive background, but only when the switch was in the ON orientation . Also, the number of fimbriae produced by a phase-locked ON strain was significantly higher in a hns-positive background . By means of competitive gel retardation and DNase I protection assays, H-NS binding to DNA segments adjacent to and within the phase switch region was demonstrated.

Mol Gen Genet, 1998 Aug, 259(3), 317 - 26
CRP down-regulates adenylate cyclase activity by reducing the level of phosphorylated IIA(Glc), the glucose-specific phosphotransferase protein, in Escherichia coli; Takahashi H et al.; The cellular cAMP level is markedly down-regulated by cAMP receptor protein (CRP) in Escherichia coli . CRP regulates adenylate cyclase both at the level of transcription of its structural gene cya and at the level of enzyme activity . We established a method to determine the phosphorylation state of IIA(Glc), the glucose-specific phosphotransferase protein, in intact cells . We found that IIA(Glc) exists predominantly in the unphosphorylated form in wild-type cells growing in LB medium, while it is largely phosphorylated in crp or cya cells . Disruption of the ptsG gene that codes for the membrane component of the major glucose transporter (IICB(Glc)), and/or the fruF gene coding for FPr (fructose-specific hybrid phosphotransferase protein), did not affect the phosphorylation state of IIA(Glc) . When IICB(Glc) was overproduced in the presence of glucose, the levels of both cAMP and phosphorylated IIA(Glc) in crp cells were concomitantly decreased to wild-type levels . In addition, when His-90 in IIA(Glc) was replaced by glutamine, both phosphorylation of IIA(Glc) and the overproduction of cAMP in crp cells were eliminated . We also found that extracts of crp+ cells markedly stimulate dephosphorylation of IIA(Glc)-P in vitro . We conclude that CRP-cAMP down-regulates adenylate cyclase primarily by reducing the level of phosphorylated IIA(Glc) . The data suggest that unspecified proteins whose expression is under the control of CRP-cAMP are responsible for this regulation.

Am J Trop Med Hyg, 1998 Sep, 59(3), 347 - 51
Development of monoclonal antibodies specifically recognizing the cyst stage of Entamoeba histolytica; Walderich B et al.; Protozoan cysts were isolated according to a two-step sucrose gradient procedure . Pure cysts of Entamoeba histolytica, in fixed and native states, were injected into BALB/c mice intraperitoneally for immunization . The spleens of these animals were used for fusion with AG8 mouse myeloma cells . Hybridomas were obtained and tested for the recognition of E . histolytica, E . dispar, E . coli, E . hartmanni, Endolimax nana, Jodamoeba biitschlii, and Giardia lamblia . Three monoclonal antibodies were identified that reacted only with cysts and trophozoites of E . histolytica . These can be used for differentiation and identification of E . histolytica in feces.

Mol Cells, 1998 Aug 31, 8(4), 483 - 90
Transcriptional control of the glnD gene is not dependent on nitrogen availability in Escherichia coli; Kim IH et al.; Glutamine synthetase (GS) is one of the most important enzymes in the assimilation of nitrogenous compounds in Escherichia coli and related bacteria . For the control of its activity and biosynthesis, tricyclic cascades of uridylylation/deuridylylation of PII protein, adenylylation/deadenylylation of glutamine synthetase, and phosphorylation/dephosphorylation of Ntr1 are operating, where the regulation of uridylylation/deuridylylation by uridylyl transferase-uridylyl removing enzyme (UT-UR) (the product of the glnD gene) would play the ultimate nitrogen sensing role . However, the possible nitrogen-regulatable element in the upstream of the glnD gene has been debated . In the present experiment, we have cloned and sequenced the four minute regions of the Escherichia coli chromosome, where rpsB, map, glnD, and dapD genes have been identified in sequence . We could localize the transcriptional start site at seven nucleotides upstream of the translation initiation codon by primer extension analysis . The nitrogen dependency of the glnD gene has been analyzed by Northern blot, RNase protection, and promoter-luciferase activity assays . These data suggested a constitutive expression of the glnD gene independent of nitrogen availability . From these results, it could be concluded that the ultimate nitrogen sensing device for the bacteria should be the UT-UR itself, through modulation of its activity in response to the nitrogen status rather than its biosynthetic mechanism.

Mol Cells, 1998 Aug 31, 8(4), 478 - 82
Expression, purification, and characterization of a familial amyotrophic lateral sclerosis-associated D90A Cu,Zn-superoxide dismutase mutant; Kim SM et al.; Cu,Zn-superoxide dismutase (SOD) is known to be a locus of mutation in familial amyotrophic lateral sclerosis (FALS) . We cloned the FALS mutant, D90A, and wild-type of human Cu,Zn-SOD, overexpressed them in E . coli, purified the proteins, and studied their properties . We investigated their enzymic activities for catalyzing the dismutation of superoxide anions and the generation of free radicals with H2O2 as a substrate . Our results showed that both wild-type and mutant enzymes have identical dismutation activities . However, the hydroxyl radical-generating function of the D90A mutant, as measured using a 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate), was enhanced relative to that of the wild-type enzyme . Catalysis of this reaction by D90A was more sensitive to inhibition by the copper chelators, penicillamine and diethyldithiocarbamate, than was catalysis by wild-type Cu,Zn-SOD . Our study suggests that this gain-of-function of FALS mutant may, in part, be responsible for the development of FALS symptoms.

Mol Cells, 1998 Aug 31, 8(4), 438 - 43
Cloning, nucleotide sequence, and expression of the DNA ligase-encoding gene from Thermus filiformis; Kim HK et al.; The gene encoding Thermus filiformis (Tfi) DNA ligase was cloned and its nucleotide sequence was determined by the chain-termination method . The primary structure of Tfi DNA ligase was deduced from its nucleotide sequence . The Tfi DNA ligase comprises of 667 amino acid residues and its molecular mass was determined to be 75,936 Da . The deduced amino acid sequence of Tfi DNA ligase showed a 86.5% homology to Tth DNA ligase and 43.5% to E . coli DNA ligase . The Lys-116 of Lys-Val-Asp-Gly motif was proposed to be the active residue of Tfi DNA ligase . In comparison with the amino acid composition of DNA ligase, Tfi DNA ligase showed a significant increase in the proportion of charged residues, Arg and Glu, compared to E . coli DNA ligase . The G + C content in the first, second, and third positions of the codons used were 70.3%, 40.3%, and 90.3%, respectively . Codon usage in Tfi DNA ligase was heavily biased towards the use of G + C in the third position . Under tac promoter control, Tfi DNA ligase was overproduced to greater than 9% of E . coli BL26Blue cellular proteins.

Mol Cells, 1998 Aug 31, 8(4), 416 - 23
Overexpression and characterization of the cDNA encoding the coat protein of cucumber mosaic virus (Strain ABI) isolated in Korea; Kim YM et al.; We constructed a recombinant CMVCP expression vector termed pMALCMV in which cDNA fragment encoding CMVCP is ligated into pMAL-c2, an E . coli expression vector . Overexpression of pMALCMV containing the entire open reading frame of CMV cDNA sequence and the maltose binding protein (MBP) leader gene was facilitated in E . coli TB1 cells, which resulted in the production of a fusion protein of MBP-CMVCP (Mr 67.7 kDa) that was immunoprecipitable with rabbit polyclonal antiserum specific for MBP . The CMVCP (Mr 24.5 kDa) was isolated through a preparative SDS polyacrylamide gel following digestion of the affinity ligand purified fusion protein with Factor Xa . The partial amino acid sequences of the cleaved proteins were confirmed at the amino terminus by peptide sequencing . The CMVCP antiserum was also prepared by intraperitoneal injection of this purified CP into a BALB/c mouse . Immunoblot analysis showed that the purified CMVCP from the Factor Xa cleavage reaction was an authentic overexpression product of the cloned CMVCP . Using an RNA mobility shift assay, it was demonstrated that CMVCP can bind to its own RNA transcript in a concentration dependent manner . However, the complex formed between CMVCP and its RNA was abolished by the addition of a polyclonal antibody that had been raised against CMVCP, confirming that the overexpressed CMVCP specifically interacts with its own RNA . Thus, our results can provide a basis for the development of a hybridoma cell line expressing the monoclonal antibody for CMVCP and molecular cloning of their genes, which may lead to the creation of CMV-resistant transgenic plants.

J Dairy Sci, 1998 Aug, 81(8), 2159 - 64
Influence of route of vaccine administration against experimental intramammary infection caused by Escherichia coli; Tomita GM et al.; The route of immunization of a commercially available Escherichia coli J5 bacterin was investigated . Jersey cows were randomly assigned to one of three treatment groups: 1) unvaccinated (control), 2) vaccinated subcutaneously in the neck, and 3) vaccinated in the area of the supramammary lymph node . Cows were vaccinated at drying off and at 2 wk prior to anticipated calving . Two quarters of each cow were challenged with approximately 60 cfu of E . coli at 14 d postcalving . Route of immunization in the neck or the area of the supramammary lymph node did not influence severity of coliform mastitis . However, the mean number of colony-forming units of E . coli recovered from challenged quarters was significantly lower for vaccinated cows than for control cows at 24 h postchallenge . A quicker milk yield recovery following intramammary challenge was also observed for vaccinated cows . Serum immunoglobulin (Ig) G, IgG1, and IgG2 and whey IgG1 and IgG2 antibody titers against E . coli J5 whole-cell antigens were significantly enhanced in vaccinated cows . Somatic cell counts in milk from challenged quarters and rectal temperatures following intramammary challenge were not different for cows across treatment groups . Immunization did not prevent intramammary infection.

Genetika, 1998 Jul, 34(7), 1017 - 20
{Eukaryotic DNA topoisomerase and in vitro recombination between circular supercoiled plasmid DNA's in an in vitro system}; Glazkov MV et al.; Eukaryotic DNA topoisomerase II was shown to catalyze recombination between circular supercoiled plasmid DNAs in vitro . The results obtained are discussed with regard to the organization of eukaryotic chromosomes in the form of topologically independent domains (loops).

Bioorg Khim, 1998 Jul, 24(7), 530 - 8
{Regulation of synthesis of ribosomal protein L7-L12: role of intercistronic rplJL region as an enhancer of translation}; Artamonova VS et al.; The ability of the Escherichia coli intercistronic rplJL region to initiate effectively the synthesis of the ribosomal protein L7/12, the only ribosomal component present in the ribosome in four copies rather than in one was studied in vivo and in vitro . It was shown that the structural determinants located upstream from the Shine-Dalgarno sequence and sharing structural motifs with the known E . coli translational enhancers are necessary for high activity of this region in translation initiation . These data indicate that mRNA-protein interactions through the ribosomal S1 protein play an important role in the formation of the initiation complex, and an enhancer region within the leader of the L7/12 mRNA serves as a target for this protein.

Bioorg Khim, 1998 Jul, 24(7), 523 - 9
{Production of recombinant human keratinocyte growth factor in Escherichia coli cells}; Ptitsyn LR et al.; Here we describe the synthesis of the gene encoding human keratinocyte growth factor (hKGF) and the construction of vectors for cytoplasmic production of hKGF in Escherichia coli cells . The level of recombinant protein expression was 1-1.5% of the total cell protein . hKGF was purified to homogeneity by three-step ion-exchange and affinity chromatography . The protein is biologically active: it stimulates dose-dependent protein synthesis in cultured human epidermal keratinocytes.

Biochim Biophys Acta, 1998 Sep 8, 1387(1-2), 462 - 8
Carbamoyl-phosphate synthetase II in kinetoplastids; Nara T et al.; Genes for carbamoyl-phosphate synthetase II (CPS II), the first enzyme of de novo pyrimidine biosynthesis, were cloned from kinetoplastids, Trypanosoma cruzi and Leishmania mexicana . T . cruzi CPS II gene encodes a protein of 1524 amino acids that encompasses the glutaminase and CPS domains, but incorporates neither aspartate carbamoyltransferase nor dihydroorotase . The residue corresponding to lysine 993 of Escherichia coli CPS, a residue that characterizes the CPS inhibited by UMP and that is replaced by tryptophan in those inhibited by UTP, is in kinetoplastids a hydrophilic glutamine, in line with the preferential inhibition by UDP of kinetoplastid CPS II.

Biochim Biophys Acta, 1998 Sep 8, 1387(1-2), 422 - 32
Protein trans-splicing and functional mini-inteins of a cyanobacterial dnaB intein; Wu H et al.; A 429 aa theoretical intein is encoded in the dnaB gene (DNA helicase) of the cyanobacterium Synechocystis sp . strain PCC6803 . This intein is shown to be capable of protein splicing with or without its native exteins when tested in E . coli cells . A centrally located 275 amino acid sequence (residues 107-381) of this intein can be deleted without loss of the protein splicing activity, resulting in a functional mini-intein of 154 aa in size . Efficient in vivo protein trans-splicing was observed when this mini-intein was split into a 106 aa N-terminal fragment containing intein motifs A and B, and a 48 aa C-terminal fragment containing intein motifs F and G . These results indicate that the N- and C-terminal regions of the Ssp DnaB intein, whether covalently linked with each other or not, can come together through non-covalent interaction to form a protein splicing domain that is functionally sufficient and structurally independent from the centrally located endonuclease domain of the intein.

Biochim Biophys Acta, 1998 Sep 8, 1387(1-2), 387 - 94
Phosphorylation of Mycobacterium leprae heat-shock 70 protein at threonine 175 alters its substrate binding characteristics; Peake P et al.; We have examined the functional properties including autophosphorylation of the Mycobacterium leprae Hsp70 homologue . Recombinant M . leprae Hsp70 had pH optima for its adenosine triphosphatase and autophosphorylating activities which were near pH 8 and 6, respectively . Both these activities were inhibited by reduced and alkylated bovine pancreatic trypsin inhibitor, but not other tested substrates . Autophosphorylation was augmented by up to 25 mM Ca2+ . Using site-directed mutagenesis to construct two Thr-->Ala mutants at positions 175 and 193, and phosphoamino acid analysis, it was shown that Thr175 was the dominant threonine residue autophosphorylated in M . leprae Hsp70 . Phosphorylation led to an increased affinity for a model polypeptide substrate, reduced and alkylated bovine albumin . These properties are compared with those of the DnaK protein of Escherichia coli.

Biochim Biophys Acta, 1998 Aug 28, 1393(2-3), 267 - 76
A phospholipase A2 is transiently synthesized during seed germination and localized to lipid bodies; May C et al.; A patatin-like protein is present in the storage tissue of cucumber seedlings during the stage of fat mobilization . The cucumber protein is a homologue of a glycoprotein which in potatoes accounts for most of the total protein content of tubers . Following preparation of a cucumber cDNA library representing the developmental stage of cotyledons of 1 day old germinating seeds we isolated and characterized a clone encoding a patatin-like protein . Antibodies raised against the protein expressed in bacteria were used for immunodetection in subcellular fractions of cucumber seedlings . It was shown that the patatin-like protein was virtually exclusively confined to lipid bodies . The protein expressed in bacteria was characterized in vitro by its esterase activity acting on monoacylglycerols and phospholipids . Detailed analysis using various forms of phosphatidyl choline as substrates demonstrated that the patatin-like protein is a phospholipase A2 acting on palmitoyl, linoleoyl and hydroperoxidized linoleoyl groups equally well . Studying the temporal and tissue-specific expression of patatin-like protein mRNA we showed its appearance exclusively during fat catabolism . As maximal amounts of the protein were found at an early stage of mobilization and confined to lipid bodies, we propose that the patatin-like hydrolase is involved in lipid body mobilization.

Biochim Biophys Acta, 1998 Sep 8, 1387(1-2), 136 - 42
The role of tryptophan residues in Escherichia coli arginyl-tRNA synthetase; Zhang QS et al.; The effect of N-bromosuccinimide (NBS) on the activity of Escherichia coli arginyl-tRNA synthetase (ArgRS) was studied . The results showed that only one tryptophan residue was easy of access to the reagent and was closely related to enzyme activity . When all the five tryptophan residues in ArgRS were changed via site-directed mutagenesis singly into Ala, the aminoacylation activity of the Trp162 mutated enzyme decreased seriously, while the other four mutant enzymes retained almost the same activity as the native one . The oxidation of the five mutant enzymes with NBS demonstrated that only the mutation of Trp162 resulted in the loss of sensitivity to the reagent . These results strongly suggest that Trp162 is more accessible to NBS and is related to enzyme activity . Furthermore, the far-UV CD spectroscopy of the mutant enzyme ArgRS162WA showed little change in its secondary structure . Finally, studies on the kinetics of the mutant enzyme ArgRS162WA in aminoacylation reaction showed that the reduction in activity could be attributed to the decrease in the values of kcat and kcat/Km for arginine . The thermodynamic calculation indicates that this mutation causes a decrease of the binding energy by 2.7 kJ/mol . Our data suggest that Trp162 is involved in the binding of arginine and in the transition state stabilization.

Mutat Res, 1998 Oct 12, 421(1), 121 - 36
A comparative study of in vivo mutation assays: analysis of hprt, lacI, cII/cI and as mutational targets for N-nitroso-N-methylurea and benzo{a}pyrene in Big Blue mice; Monroe JJ et al.; We have compared the response of the native hprt gene and the lacI, cII, and cI transgenes in Big Blue B6C3F1 mice following treatment with either N-nitroso-N-methylurea (MNU) or benzo{a}pyrene (BaP) . Three weeks after mutagen treatment splenic T cells were isolated from the animals, and samples were either cultured to measure mutation at the native hprt locus or used to extract genomic DNA for transgene mutation analysis . Phage rescued from extracted DNA were plated in the presence of 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) to score lacI mutations, or plated on a hflAB lawn to score cII and cI mutants . With MNU hprt mutant frequency increased in a dose-related, sublinear manner up to 78-fold above background at the highest dose tested (20 mg/kg) . In comparison, the lacI transgene yielded only a 3.1-fold increase at this dose, and the cII and cI transgenes did not show any increase . With 150 mg/kg BaP a 5.8- and 8.7-fold increase in mutant frequency was observed at hprt and lacI, respectively, while only a 1.3-fold increase was observed at cII . DNA sequencing revealed an increase in GC-->TA transversions among the cII mutants, suggesting that the increase was related to BaP exposure . No significant increase in cI mutant frequency was observed . Therefore, the order of mutation assay sensitivity was hprt>lacI>cII/cI with MNU, and hprt approximately lacI> cII/cI with BaP . While the hflAB selection system offers significant advantages with respect to cost and effort when compared to the lacI assay, additional evaluation of its sensitivity is warranted .

Biochim Biophys Acta, 1998 Oct 1, 1400(1-3), 29 - 43
DNA gyrase and topoisomerase IV: biochemical activities, physiological roles during chromosome replication, and drug sensitivities; Levine C et al.; DNA gyrase and topoisomerase IV are the two type II topoisomerases present in bacteria . Though clearly related, based on amino acid sequence similarity, they each play crucial, but distinct, roles in the cell . Gyrase is involved primarily in supporting nascent chain elongation during replication of the chromosome, whereas topoisomerase IV separates the topologically linked daughter chromosomes during the terminal stage of DNA replication . These different roles can be attributed to differences in the biochemical properties of the two enzymes . The biochemical activities, physiological roles, and drug sensitivities of the enzymes are reviewed.

Biochim Biophys Acta, 1998 Oct 1, 1400(1-3), 3 - 18
Structure of DNA topoisomerases; Berger JM; Over the last several years topoisomerases have finally begun to yield to high-resolution structural studies . These models have greatly aided our understanding of the mechanisms of topoisomerase catalysis and drug interactions . This review will cover advances in the structural biology of topoisomerases and discuss their implications for topoisomerase function.

J Bacteriol, 1998 Oct, 180(19), 5279 - 83
Two amino acid residues of transposase contributing to differential transposability of IS1 elements in Escherichia coli; Chen JH et al.; Escherichia coli W3110 contains four types of IS1 elements in the chromosome . Using an insertion element entrapping system, we collected 116 IS1 plasmid insertion mutants, which resulted from a minimum of 26 independent IS1 insertion events . All of them had insertions of IS1 of the IS1A (IS1E and IS1G) type . Inspection of the transposase sequences of the four IS1 types and the IS1 of the resistance plasmid R100 showed that two amino acid residues, His-193 and Leu-217 of transposase, might contribute to differential transposability of IS1 elements in W3110 . The two amino acid residues of the transposase in IS1A (IS1E and IS1G) were altered separately by site-directed mutagenesis, and each mutant was found to mediate transposition at a frequency about 30-fold lower than that of IS1A (IS1E and IS1G) . Thus, the assumption that His-193 and Leu-217 of transposase contribute to differential transposability of IS1 elements in W3110 was confirmed.

J Bacteriol, 1998 Oct, 180(19), 5243 - 6
The gene for 16S rRNA methyltransferase (ksgA) functions as a multicopy suppressor for a cold-sensitive mutant of era, an essential RAS-like GTP-binding protein in Escherichia coli; Lu Q et al.; Era, a Ras-like GTP-binding protein in Escherichia coli, has been shown to be essential for growth . However, its cellular functions still remain elusive . In this study, a genetic screening of an E . coli genomic library was performed to identify those genes which can restore the growth ability of a cold-sensitive mutant, Era(Cs) (E200K), at a restrictive temperature when expressed in a multicopy plasmid . Among eight suppressors isolated, six were located at 1 min of the E . coli genomic map, and the gene responsible for the suppression of Era(Cs) (E200K) was identified as the ksgA gene for 16S rRNA transmethylase, whose mutation causes a phenotype of resistance to kasugamycin, a translation initiation inhibitor . This is the first demonstration of suppression of impaired function of Era by overproduction of a functional enzyme . A possible mechanism of the suppression of the Era cold-sensitive phenotype by KsgA overproduction is discussed.

J Bacteriol, 1998 Oct, 180(19), 5240 - 2
Regulation of Escherichia coli secA by cellular protein secretion proficiency requires an intact gene X signal sequence and an active translocon; Oliver D et al.; secA is translationally regulated by the protein secretion proficiency state of the Escherichia coli cell . This regulation was explored by making signal sequence mutations in the gene upstream of secA, gene X, which promotes secA translational coupling . Gene X signal sequence mutants were constitutive for secA expression, while prlA alleles partially restored secA regulation . These results show that interaction of the pre-gene X protein with the translocon is required for proper secA regulation . Furthermore, gene X signal sequence mutations disrupted secA regulation only in the cis configuration . We propose that nascent pre-gene X protein interacts with the translocon during its secretion to constitute the secretion sensor.

J Bacteriol, 1998 Oct, 180(19), 5173 - 82
ClpB in a cyanobacterium: predicted structure, phylogenetic relationships, and regulation by light and temperature; Celerin M et al.; The sequence of a genomic clone encoding a 100-kDa stress protein of Plectonema boryanum (p-ClpB) was determined . The predicted polypeptide contains two putative ATPase regions located within two highly conserved domains (N1 and N2), a spacer region that likely forms a coiled-coil domain, and a highly conserved consensus CK2 phosphorylation domain . The coiled-coil region and the putative site of phosphorylation are not unique to p-ClpB; they are present in all ClpB sequences examined and are absent from the ClpB paralogs ClpA, ClpC, ClpX, and ClpY . Small quantities of a 4.5-kb p-clpB transcript and 110-kDa cytosolic p-ClpB protein were detected in cells grown under optimal conditions; however, increases in the quantities of the transcript and protein were observed in cells grown under excess light and low temperature conditions . Finally, we analyzed ClpA, ClpB, and ClpC sequences from 27 organisms in order to predict phylogenetic relationships among the homologs . We have used this information, along with an identity alignment, to redefine the Clp subfamilies.

J Bacteriol, 1998 Oct, 180(19), 5165 - 72
Roles of the Escherichia coli small heat shock proteins IbpA and IbpB in thermal stress management: comparison with ClpA, ClpB, and HtpG In vivo; Thomas JG et al.; We have constructed an Escherichia coli strain lacking the small heat shock proteins IbpA and IbpB and compared its growth and viability at high temperatures to those of isogenic cells containing null mutations in the clpA, clpB, or htpG gene . All mutants exhibited growth defects at 46 degrees C, but not at lower temperatures . However, the clpA, htpG, and ibp null mutations did not reduce cell viability at 50 degrees C . When cultures were allowed to recover from transient exposure to 50 degrees C, all mutations except Deltaibp led to suboptimal growth as the recovery temperature was raised . Deletion of the heat shock genes clpB and htpG resulted in growth defects at 42 degrees C when combined with the dnaK756 or groES30 alleles, while the Deltaibp mutation had a detrimental effect only on the growth of dnaK756 mutants . Neither the overexpression of these heat shock proteins nor that of ClpA could restore the growth of dnaK756 or groES30 cells at high temperatures . Whereas increased levels of host protein aggregation were observed in dnaK756 and groES30 mutants at 46 degreesC compared to wild-type cells, none of the null mutations had a similar effect . These results show that the highly conserved E . coli small heat shock proteins are dispensable and that their deletion results in only modest effects on growth and viability at high temperatures . Our data also suggest that ClpB, HtpG, and IbpA and -B cooperate with the major E . coli chaperone systems in vivo.

J Bacteriol, 1998 Oct, 180(19), 5123 - 8
CheZ has no effect on flagellar motors activated by CheY13DK106YW; Scharf BE et al.; The behaviors of both cheZ-deleted and wild-type cells of Escherichia coli were found to be very sensitive to the level of expression of CheZ, a protein known to accelerate the dephosphorylation of the response regulator CheY-phosphate (CheY-P) . However, cells induced to run and tumble by the unphosphorylated mutant protein CheY13DK106YW (CheY**) failed to respond to CheZ, even when CheZ was expressed at high levels . Therefore, CheZ neither affects the flagellar motors directly nor sequesters CheY** . In in vitro cross-linking studies, CheY** promoted trimerization of CheZ to the same extent as wild-type CheY but failed to induce the formation of complexes of higher molecular weight observed with CheY-P . Also, CheY** could be cross-linked to FliM, the motor receptor protein, nearly as well as CheY-P . Thus, to CheZ, CheY** looks like CheY, but to FliM, it looks like CheY-P.

J Bacteriol, 1998 Oct, 180(19), 5109 - 16
Effect of slow growth on metabolism of Escherichia coli, as revealed by global metabolite pool ("metabolome") analysis; Tweeddale H et al.; Escherichia coli growing on glucose in minimal medium controls its metabolite pools in response to environmental conditions . The extent of pool changes was followed through two-dimensional thin-layer chromatography of all 14C-glucose labelled compounds extracted from bacteria . The patterns of metabolites and spot intensities detected by phosphorimaging were found to reproducibly differ depending on culture conditions . Clear trends were apparent in the pool sizes of several of the 70 most abundant metabolites extracted from bacteria growing in glucose-limited chemostats at different growth rates . The pools of glutamate, aspartate, trehalose, and adenosine as well as UDP-sugars and putrescine changed markedly . The data on pools observed by two-dimensional thin-layer chromatography were confirmed for amino acids by independent analysis . Other unidentified metabolites also displayed different spot intensities under various conditions, with four trend patterns depending on growth rate . As RpoS controls a number of metabolic genes in response to nutrient limitation, an rpoS mutant was also analyzed for metabolite pools . The mutant had altered metabolite profiles, but only some of the changes at slow growth rates were ascribable to the known control of metabolic genes by RpoS . These results indicate that total metabolite pool ("metabolome") analysis offers a means of revealing novel aspects of cellular metabolism and global regulation.

J Bacteriol, 1998 Oct, 180(19), 5102 - 8
Expression of the Kdp ATPase is consistent with regulation by turgor pressure; Malli R et al.; The kdpFABC operon of Escherichia coli encodes the four protein subunits of the Kdp K+ transport system . Kdp is expressed when growth is limited by the availability of K+ . Expression of Kdp is dependent on the products of the adjacent kdpDE operon, which encodes a pair of two-component regulators . Studies with kdp-lac fusions led to the suggestion that change in turgor pressure acts as the signal to express Kdp (L . A . Laimins, D . B . Rhoads, and W . Epstein, Proc . Natl . Acad . Sci . USA 78:464-468, 1981) . More recently, effects of compatible solutes, among others, have been interpreted as inconsistent with the turgor model (H . Asha and J . Gowrishankar, J . Bacteriol . 175:4528-4537, 1993) . We re-examined the effects of compatible solutes and of medium pH on expression of Kdp in studies in which growth rate was also measured . In all cases, Kdp expression correlated with the K+ concentration when growth began to slow . Making the reasonable but currently untestable assumptions that the reduction in growth rate by K+ limitation is due to a reduction in turgor and that addition of betaine does not increase turgor, we concluded that all of the data on Kdp expression are consistent with control by turgor pressure.

Biochemistry, 1998 Sep 22, 37(38), 13269 - 75
Ligand-induced conformational transitions in Escherichia coli phosphofructokinase 2: evidence for an allosteric site for MgATP2-; Guixe V et al.; The binding of ligands to phosphofructokinase 2 (Pfk-2) from Escherichia coli induces changes in the fluorescence emission properties of its single tryptophan residue, Trp88, suggesting that upon binding the protein undergoes a conformational change . This fluorescence probe was used to determine the presence of an allosteric site for MgATP2- in the enzyme . Fructose 6-phosphate (fructose-6-P), the first substrate that binds to the enzyme with an ordered bi-bi mechanism, increases the fluorescence up to 30% . The saturation curve for this compound is hyperbolic with a Kd of 6 microM . The titration of Pfk-2 with MgATP2- causes a quenching of fluorescence of about 30% of its initial value, with a blue shift of 7 nm in the emission maximum . The response is cooperative with a Kd of 80 microM and a Hill coefficient of 2 . The interaction of MgATP2- cannot take place at the active site in the absence of fructose-6-P, due to the ordered kinetic mechanism . Addition of compounds that bind to the catalytic site of Pfk-2, such as ATP4- or Mg-AMP-PNP, did not produce significant changes in the fluorescence spectrum of Trp88 . However, in the absence of Mg2+, the addition of ATP4- to the enzyme-fructose-6-P complex shows a hyperbolic increase of fluorescence of 8% . Acrylamide steady-state quenching experiments for different enzyme-ligand complexes of Pfk-2, indicate that the tryptophan in the enzyme-MgATP2- complex is exposed to a much smaller extent to the solvent than in the free enzyme or in the enzyme-fructose-6-P complex . The effect of the binding of fructose-6-P or MgATP2- on the polarization fluorescence of the emission of Trp88 in Pfk-2 indicates changes in the local mobility of the Trp88 in both enzyme complexes . The average lifetime for Trp88 in Pfk-2 was found to be unusually large, approximately 7.7 ns, and did not vary significantly with the ligation state of the enzyme, which demonstrates that the quenching or enhancement of fluorescence induced by the ligands is mainly due to the complex formation with Pfk-2 . These results demonstrate the presence of an allosteric site for MgATP2- in Pfk-2 from E . coli, responsible for the inhibition of the enzyme activity by this ligand.

Biochemistry, 1998 Sep 22, 37(38), 13230 - 8
Kinetic mechanism of Escherichia coli asparagine synthetase B; Boehlein SK et al.; Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartate, glutamine, and ATP . A combination of kinetic, isotopic-labeling, and stoichiometry studies have been performed to define the nature of nitrogen transfer mediated by AS-B . The results of initial rate studies were consistent with initial binding and hydrolysis of glutamine to glutamate plus enzyme-bound ammonia . The initial velocity results were equally consistent with initial binding of ATP and aspartate prior to glutamine binding . However, product inhibition studies were only consistent with the latter pathway . Moreover, isotope-trapping studies confirmed that the enzyme-ATP-aspartate complex was kinetically competent . Studies using 18O-labeled aspartate were consistent with formation of a beta-aspartyl-AMP intermediate, and stoichiometry studies revealed that 1 equiv of this intermediate formed on the enzyme in the absence of a nitrogen source . Taken together, our results are most consistent with initial formation of beta -aspartyl-AMP intermediate prior to glutamine binding . This sequence leaves open many possibilities for the chemical mechanism of nitrogen transfer.

Biochemistry, 1998 Sep 22, 37(38), 13212 - 21
Allosteric activation of Arabidopsis threonine synthase by S-adenosylmethionine; Curien G et al.; Plant threonine synthase, in contrast to its bacterial counterpart, is strongly stimulated by S-adenosylmethionine via a noncovalent interaction {Giovanelli et al . (1984) Plant . Physiol . 76, 285-292} . The mechanism of activation remained, however, largely unknown . To further characterize this unusual role for S-adenosylmethionine, the Arabidopsis thaliana threonine synthase was overexpressed in Escherichia coli, purified to homogeneity, and then used for kinetic and enzyme-bound pyridoxal 5'-phosphate fluorescence equilibrium-binding experiments . We observed that the activating effect of S-adenosylmethionine results from an 8-fold increase in the rate of catalysis and from a 25-fold decrease in the Km value for the O-phosphohomoserine substrate . The data can be well fitted by a kinetic model assuming binding of two S-adenosylmethionine molecules on the native enzyme . We suggest that the dramatic modifications of the enzyme kinetic properties originate most presumably from an allosteric and cooperative transition induced by S-adenosylmethionine . This transition occurs at a much faster rate in the presence of the substrate than in its absence.

World J Surg, 1998 Oct, 22(10), 1069 - 75; discussion 1076
Wound healing of intestinal anastomosis after digestive surgery under septic conditions: participation of local interleukin-6 expression; Ishimura K et al.; This study aimed to evaluate the integrity of anastomotic wound healing after digestive surgery under septic conditions and to observe local interleukin-6 (IL-6) expression around the anastomotic segment . Experimental animals were separated into lipopolysaccharide (LPS) and control groups . Each was injected with LPS or normal saline solution into the peritoneal cavity 24 hours before transection and anastomosis of the colon . The anastomotic bursting pressure (ABP) and tissue hydroxyproline concentration (HP) were measured as indicators of wound healing . Immunohistochemical staining for IL-6 was performed on tissue samples obtained from the anastomotic segment, lung, liver, and kidney . The reactive cells were counted by light microscopy . The ABP and HP were significantly lower in the LPS group than the control group 7 days after the surgery . In the LPS group, IL-6 expression around the anastomotic segment was enhanced 1 and 6 hours after surgery but suppressed 24 hours afterward . In contrast, IL-6 expression in lung, liver, and kidney was enhanced in the LPS group 24 hours after surgery but not in the control group . It is suggested that anastomotic wound healing is impaired after digestive tract surgery under septic conditions, and local IL-6 expression participates in wound healing.

J Biol Chem, 1998 Oct 2, 273(40), 26130 - 7
Cloning and characterization of the 5'-flanking region for the human topoisomerase III gene; Kim JC et al.; The human DNA topoisomerase III (hTOP3) gene encodes a topoisomerase homologous to the Escherichia coli DNA topoisomerase I subfamily . To understand the mechanisms responsible for regulating hTOP3 expression, we have cloned the 5'-flanking region of the gene coding for the hTOP3 and analyzed its promoter activity . The presence of a single transcription initiation site was suggested by primer extension analysis . The hTOP3 gene promoter is moderately high in GC content and lacks a canonical TATA box, suggesting that hTOP3 promoter has overall similarity to promoters of a number of housekeeping genes . Examination of the promoter sequence indicated the presence of four Sp-1 consensus binding sequences and a putative initiator element surrounding the transcription initiation site . Transient expression of a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the 52-base pair region from -326 to -275 upstream of the transcription initiation site includes a positive cis-acting element(s) for the efficient expression of hTOP3 gene . On the basis of gel mobility shift and supershift assays, we demonstrated that both YY1 and USF1 transcription factors can bind to the 52-base pair region . When HeLa cells were transiently transfected with a mutant construct which had disabled both YY1- and USF1-binding sites, the luciferase activity was greatly reduced, suggesting that these binding elements play a functional role in the basal activation of the hTOP3 promoter . Transfection studies with mutations that selectively impaired YY1 or USF1 binding suggested that both YY1 and USF1 function as activators in the hTOP3 promoter.

J Biol Chem, 1998 Oct 2, 273(40), 25974 - 86
Catabolism of phenylacetic acid in Escherichia coli . Characterization of a new aerobic hybrid pathway; Ferrandez A et al.; The paa cluster of Escherichia coli W involved in the aerobic catabolism of phenylacetic acid (PA) has been cloned and sequenced . It was shown to map at min 31.0 of the chromosome at the right end of the mao region responsible for the transformation of 2-phenylethylamine into PA . The 14 paa genes are organized in three transcription units: paaZ and paaABCDEFGHIJK, encoding catabolic genes; and paaXY, containing the paaX regulatory gene . The paaK gene codes for a phenylacetyl-CoA ligase that catalyzes the activation of PA to phenylacetyl-CoA (PA-CoA) . The paaABCDE gene products, which may constitute a multicomponent oxygenase, are involved in PA-CoA hydroxylation . The PaaZ protein appears to catalyze the third enzymatic step, with the paaFGHIJ gene products, which show significant similarity to fatty acid beta-oxidation enzymes, likely involved in further mineralization to Krebs cycle intermediates . Three promoters, Pz, Pa, and Px, driven the expression of genes paaZ, paaABCDEFGHIJK, and paaX, respectively, have been identified . The Pa promoter is negatively controlled by the paaX gene product . As PA-CoA is the true inducer, PaaX becomes the first regulator of an aromatic catabolic pathway that responds to a CoA derivative . The aerobic catabolism of PA in E . coli represents a novel hybrid pathway that could be a widespread way of PA catabolism in bacteria.

J Biol Chem, 1998 Oct 2, 273(40), 25893 - 902
Sequence characteristics, subcellular localization, and substrate specificity of DYRK-related kinases, a novel family of dual specificity protein kinases; Becker W et al.; DYRK1 is a dual specificity protein kinase presumably involved in brain development . Here we show that the kinase belongs to a new family of protein kinases comprising at least seven mammalian isoforms (DYRK1A, DYRK1B, DYRK1C, DYRK2, DYRK3, DYRK4A, and DYRK4B), the yeast homolog Yak1p, and the Drosophila kinase minibrain (MNB) . In rat tissues, DYRK1A is expressed ubiquitously, whereas transcripts for DYRK1B, DYRK2, DYRK3, and DYRK4 were detected predominantly in testes of adult but not prepuberal rats . By fluorescence microscopy and subcellular fractionation, a green fluorescent protein (GFP) fusion protein of DYRK1A was found to accumulate in the nucleus of transfected COS-7 and HEK293 cells, whereas GFP-DYRK2 was predominantly detected in the cytoplasm . DYRK1A exhibited a punctate pattern of GFP fluorescence inside the nucleus and was co-purified with the nuclear matrix . Analysis of GFP-DYRK1A deletion constructs showed that the nuclear localization of DYRK1A was mediated by its nuclear targeting signal (amino acids 105-139) but that its characteristic subnuclear distribution depended on additional N-terminal elements (amino acids 1-104) . When expressed in Escherichia coli, DYRK1A, DYRK2, DYRK3, MNB, and Yak1p catalyzed their autophosphorylation on tyrosine residues . The kinases differed in their substrate specificity in that DYRK2 and DYRK3, but not DYRK1A and MNB, catalyzed phosphorylation of histone H2B . The heterogeneity of their subcellular localization and substrate specificity suggests that the kinases are involved in different cellular functions.

J Biol Chem, 1998 Oct 2, 273(40), 25809 - 17
Random sequence mutagenesis and resistance to 5-fluorouridine in human thymidylate synthases; Landis DM et al.; Thymidylate synthase (TS) catalyzes the methylation of dUMP to dTMP and is the target for the widely used chemotherapeutic agent 5-fluorouracil . We used random sequence mutagenesis to replace 13 codons within the active site of TS and obtain variants that are resistant to 5-fluorodeoxyuridine (5-FdUR) . The resulting random library was selected for its ability to complement a TS-deficient Escherichia coli strain, and sequence analysis of survivors found multiple substitutions to be tolerable within the targeted region . An independent selection of the library was carried out in the presence of 5-FdUR, resulting in a more limited spectrum of mutations . One specific mutation, C199L, was observed in more than 46% of 5-FdUR-resistant clones . A 5-FdUR-resistant triple mutant, A197V/L198I/C199F, was purified to apparent homogeneity . Kinetic studies with the substrate dUMP indicate that this mutant is similar to the wild type in regards to kcat and Km values for dUMP and the cosubstrate CH2H4-folate . In contrast, equilibrium binding studies with the inhibitor, FdUMP, demonstrate that the dissociation constant (Kd) for FdUMP binding into the ternary complex was 20-fold higher than values obtained for the wild-type enzyme . This 5-FdUMP-resistant mutant, or others similarly selected, is a candidate for use in gene therapy to render susceptible normal cells resistant to the toxic effects of systemic 5-fluorouracil.

J Biol Chem, 1998 Oct 2, 273(40), 25741 - 4
Superoxide dependence of the toxicity of short chain sugars; Benov L et al.; Erythrose inhibited the growth of a sodA sodB strain of Escherichia coli under aerobiosis; but did not inhibit anaerobic growth of the sodA sodB strain, or the aerobic growth of the superoxide dismutase (SOD)-competent parental strain . A SOD mimic protected the sodA sodB strain against the toxicity of erythrose as did the carbonyl-blocking reagents hydrazine and aminoguanidine . Three carbon sugars, such as glyceraldehyde and dihydroxy acetone, and the two carbon sugar glycolaldehyde, were similarly toxic in an O-2-dependent manner . An unidentified dialyzable component in E . coli extract augmented the oxidation of short chain sugars, and this was partially inhibitable by SOD . The toxicity of the short chain sugars appears to be because of an O-2-dependent oxidation to alpha, beta-dicarbonyl compounds . In keeping with this view was the O-2-independent toxicity of methylglyoxal.

J Biol Chem, 1998 Oct 2, 273(40), 25594 - 601
Phenylalanine hydroxylase from Chromobacterium violaceum . Uncoupled oxidation of tetrahydropterin and the role of iron in hyroxylation; Chen D et al.; A gene encoding phenylalanine hydroxylase has been cloned from Chromobacterium violaceum and expressed in Escherichia coli . The purified phenylalanine hydroxylase contains copper, which does not support enzymatic activity . Upon removal of copper by dithiothreitol (DTT), the enzyme contains substoichiometric amounts of calcium and zinc but little or no redox-active metal ions . The copper-depleted hydroxylase catalyzes the phenylalanine-dependent oxidation of 6, 7-dimethyltetrahydropterin (DMPH4) by O2 in a reaction in which phenylalanine is not hydroxylated and does not appear to undergo a chemical change, and hydrogen peroxide is produced . Analogs of phenylalanine also activate the oxidation of DMPH4 . Both the copper-phenylalanine hydroxylase and the copper-depleted hydroxylase catalyze the hydroxylation of phenylalanine in the presence of DTT and FeSO4 in a reaction in which hydrogen peroxide is not produced . The apparent values of Km for Fe2+ and DTT are 0.28 microM and 1.1 mM, respectively, at 1.0 mM phenylalanine, 120 microM DMPH4 and pH 7 . 4 and 23 degreesC . The apparent value of kcat is 14.3 s-1 under these conditions . Glutathione, mercaptoethanol, and dihydrolipoate support the hydroxylation of phenylalanine essentially as well as DTT . Incubation of copper-depleted hydroxylase with FeSO4, phenylalanine, and DTT followed by gel permeation chromatography leads to an iron-hydroxylase containing approximately 1 molecule of iron per molecule of enzyme . The iron-hydroxylase displays an optical absorption band extending from 300 to 600 nm, and it catalyzes the hydroxylation of phenylalanine at the same maximum rate as the iron-activated hydroxylase but does not require added Fe2+ . We conclude that iron participates in the hydroxylation of phenylalanine . Iron is not required for the oxidation of DMPH4, although it may exert a modest acceleration effect . A hypothetical mechanism is presented wherein the reaction of iron with the putative 4a-hydroperoxy-DMPH4 leads to 4a-hydroxy-DMPH4 and a high valent iron-oxy species . The iron-oxy species is postulated to react with phenylalanine in the hydroxylation process.

Plant Mol Biol, 1998 Oct, 38(3), 481 - 90
Tissue- and stage-specific expression of a soybean (Glycine max L.) seed-maturation, biotinylated protein; Hsing YC et al.; A cDNA clone GmPM4 which encodes mRNA species in mature or dry soybean seeds was characterized . DNA sequence analysis shows that the deduced polypeptides have a molecular mass of 68 kDa . GmPM4 proteins have a relatively high amino acid sequence homology with a major biotinylated protein isolated from pea seeds, SBP65, but both of these proteins differ markedly from that of presently known biotin enzymes . The accumulation of GmPM4 mRNA is detectable in the leaf primodium and the vascular tissues of the hypocotyl-radicle axis of mature seeds, and the GmPM4 proteins are present at high levels in dry and mature soybean seeds, but not in fresh immature seeds . It degrades rapidly at the early stage of seed germination . These proteins are boiling-soluble and biotinylated when they are present endogenously in soybean seeds; however, the same recombinant protein expressed in Escherichia coli is boiling-soluble, but it is not biotinylated.

Plant Mol Biol, 1998 Oct, 38(3), 347 - 56
The sugarcane bacilliform badnavirus promoter is active in both monocots and dicots; Tzafrir I et al.; Regions of the sugarcane bacilliform badnavirus genome were tested for promoter activity . The genomic region spanning nucleotides 5999-7420 was shown to possess promoter activity as exemplified by its ability to drive the expression of the coding region of the uidA gene of Escherichia coli, in both Avena sativa and Arabidopsis thaliana . In A . sativa, the promoter was active in all organs examined and, with the exception of the anthers where the expression was localized, this activity was constitutive . In A . thaliana, the promoter activity was constitutive in the rosette leaf, stem, stamen, and root and limited primarily to vascular tissue in the sepal and the silique . The transgene was inherited and active in progeny plants of both A . sativa and A . thaliana.

Plant Mol Biol, 1998 Nov 1, 38(4), 513 - 20
Substrate profiles and expression of caffeoyl coenzyme A and caffeic acid O-methyltransferases in secondary xylem of aspen during seasonal development; Meng H et al.; Seasonal expression of caffeoyl-CoA O-methyltransferase (EC 2.1.1.104) was analyzed in aspen developing secondary xylem in parallel with caffeate O-methyltransferase (EC 2.1.1.68) . Enzyme activity and mRNA levels for both enzymes peaked in the middle of the growing season . These results strongly suggest that both forms of O-methyltransferase were actively participating in lignin precursor biosynthesis during the growing season . To determine the role of each enzyme form, xylem extracts from two days in the growing season were assayed with four substrates: caffeoyl-CoA, 5-hydroxyferuloyl-CoA, caffeate acid and 5-hydroxyferulic acid . Recombinant forms of caffeoyl-CoA and caffeate O-methyltransferase were also assayed with these substrates . The recombinant enzymes have different substrate specificity with the caffeoyl-CoA O-methyltransferase being essentially specific for CoA ester substrates with a preference for caffeoyl-CoA, while caffeate O-methyltransferase utilized all four substrates with a preference for the free acid forms . We suggest that caffeoyl-CoA O-methyltransferase is likely to be responsible for biosynthesis of lignin precursors in the guaiacyl pathway and may represent a more primitive enzyme form leftover from very early land plant evolution . Caffeate O-methyltransferase is more likely to be responsible for lignin precursor biosynthesis in the syringyl pathway, especially since it can catalyze methylation of 5-hydroxyferuloyl-CoA quite effectively . This latter enzyme form then may be considered a more recently evolved component of the lignin biosynthetic pathways of the evolutionarily advanced plants such as angiosperms.

J Gen Virol, 1998 Sep, 79 ( Pt 9), 2275 - 81
cDNA cloning and molecular characterization of cherry green ring mottle virus; Zhang YP et al.; The complete nucleotide sequence of the cherry green ring mottle virus (CGRMV) genome was determined to be 8372 nt excluding a 3' poly(A) tail . Based on computer analysis and sequence comparison, five open reading frames (ORFs) were identified on the virion strand encoding: a putative RNA-dependent RNA polymerase, a triple gene block and a coat protein . Two other ORFs with Mr values over 10,000 and internal to the helicase and coat protein genes, but of unknown function, were also identified . Sequence and genome structure comparisons with other filamentous viruses indicated that CGRMV is most similar to apple stem pitting virus, some carlaviruses and potexviruses . However, it is different from members of any of these virus groups in regard to sequence homology and genome organization . A chimeric fusion coat protein was expressed in E . coli and antibodies specific for the CGRMV coat protein were raised in rabbits . The antibody was used in Western blot analyses to detect the CGRMV coat protein in infected cherry tissue.

Phytochemistry, 1998 Sep, 49(2), 319 - 25
Purification and immunological characterization of a recombinant trimethylflavonol 3'-O-methyltransferase; Seguin J et al.; A flavonol O-methyltransferase cDNA clone (pF3'OMT) from Chrysosplenium americanum was expressed in Escherichia coli Top 10 and the recombinant protein was purified to near homogeneity by affinity chromatography on chelation resin and gel filtration on Superose 12 columns . The purified protein was enzymatically active as a 42 kDa monomer and exhibited strict specificity for position 3' of 3,7,4'-trimethylquercetin . In did not accept the mono- or dimethyl analogs, the parent aglycone quercetin or the phenylpropanoids, caffeic and 5-hydroxyferulic acids as substrates; thus indicating its involvement in the later steps of polymethylated flavonol synthesis in this plant . The K(m) values of the enzyme for 3,7,4'-trimethylquercetin as substrate and S-adenosyl-L-methionine as co-substrate were 7.2 and 20 microM, respectively . The enzyme activity was strongly inhibited by both Ni2+ and rho-chloromercuribenzoate and was restored by the addition of EDTA or beta-mercaptoethanol, respectively . Antibodies raised against the F3'OMT recombinant protein recognized a protein band migrating at the expected molecular mass of the enzyme on SDS-polyacrylamide gels of protein extracts prepared from various sources . This implies a high degree of structural similarity among these enzymes that is also corroborated by their hydropathy profiles.

Gene Ther, 1998 Jun, 5(6), 809 - 19
Pre-existing herpes simplex virus 1 (HSV-1) immunity decreases, but does not abolish, gene transfer to experimental brain tumors by a HSV-1 vector; Herrlinger U et al.; The influence of pre-existing anti-herpes simplex type 1 (HSV-1) immunity on HSV-1 vector-mediated gene transfer to glioma cells was analyzed in this gene marking study using intracranial D74 gliomas in syngeneic Fischer rats . The HSV-1 mutant virus used, hrR3, is defective in ribonucleotide reductase and bears the marker genes E . coli lacZ and HSV-1 thymidine kinase (HSVtk) . Initial marker gene expression in tumors 12 h after direct virus injection was reduced in immunized animals to about 15% of that in nonimmunized animals . Marker gene expression in both sets stayed at initial levels for 2 days after intratumoral injection and declined markedly on day 5 . Inflammatory infiltrates in the tumor were more prominent in HSV-1-immunized, as compared with nonimmunized animals, at 12 and 24 h, but appeared similar at 2-5 days after injection . By day 10, the immune reaction had subsided in immunized animals and macrophages remained only in nonimmunized animals . In conclusion, gene transfer to brain tumors using a HSV-1 vector was greatly reduced, but not completely abolished, under pre-immunization conditions . Pre-existing antibodies to HSV-1 may also serve a positive role in providing an increased margin of safety in intracranial application of HSV-1 vectors by limiting spread of the virus within the brain and to other tissues.

Infect Immun, 1998 Oct, 66(10), 5031 - 5
Pathogenicity of an enterotoxigenic Escherichia coli hemolysin (hlyA) mutant in gnotobiotic piglets; Moxley RA et al.; Pigs infected with hemolytic F4(+) strains of enterotoxigenic Escherichia coli often develop septicemia secondary to intestinal infection . We tested the hypothesis that inactivation of hemolysin would reduce the ability of F4(+) enterotoxigenic E . coli to cause septicemia in swine following oral inoculation . Inactivation of the hemolysin structural gene (hlyA) did not decrease the incidence of septicemia in the gnotobiotic piglet model.

Am J Physiol, 1998 Oct, 275(4 Pt 2), H1148 - 57
Nitric oxide-independent activation of soluble guanylyl cyclase contributes to endotoxin shock in rats; Wu CC et al.; We investigated whether a complete inhibition of nitric oxide (NO) formation caused by bacterial endotoxin (lipopolysaccharide, LPS) in vivo prevents the hypotension and restores the vascular hyporeactivity to normal in vivo and ex vivo . The combination of dexamethasone (Dex; 3 mg/kg at 30 min before LPS) plus aminoguanidine (AG; 15 mg/kg at 2 h after LPS) inhibited the overproduction of nitrate (an indicator of NO) in the plasma and aortic smooth muscle and also prevented the development of the delayed hypotension in rats treated with LPS for 6 h . However, the vascular hyporeactivity to norepinephrine (NE) was only partially improved either in vivo or ex vivo in endotoxemic rats treated with Dex plus AG . Pretreatment of aortic rings with Nomega-nitro-L-arginine methyl ester (L-NAME) or 1H-{1,2, 4}oxidazolo{4,3-a}quinoxalin-1-one (ODQ) enhanced the contraction to NE in rings obtained from LPS-treated rats, but not in those from Dex plus AG-treated endotoxemic rats . Methylene blue, an inhibitor of soluble guanylyl cyclase (GC), completely restored contractions to NE and aortic cGMP levels to normal either in LPS-treated rats or in Dex plus AG-treated endotoxemic rats, whereas the cGMP level was partially inhibited by ODQ in LPS-treated rats only . These results suggest that non-NO mediator(s) also activates soluble GC during endotoxemia . Interestingly, we found that in the presence of tetraethylammonium (an inhibitor of K+ channels) plus L-NAME or charybdotoxin {a specific inhibitor of large-conductance Ca2+-activated K+ (KCa) channels} plus ODQ, the vascular hyporeactivity to NE in the LPS-treated group was also completely restored to normal . In addition, in the presence of L-NAME or ODQ, the vascular hyporeactivity to high K+ was abolished in rings from the LPS-treated group . These results suggest that LPS causes the production of other mediator(s), in addition to NO, which also stimulates soluble GC (i.e., increases the formation of cGMP) and then activates the large-conductance KCa channels in the vascular smooth muscle causing vascular hyporeactivity.

Am J Physiol, 1998 Oct, 275(4 Pt 2), H1122 - 9
Induction and cDNA sequence of inducible nitric oxide synthase from canine aortic smooth muscle cells; Wang X et al.; An inducible isoform of nitric oxide synthase (type II, iNOS) is expressed in cardiac and vascular smooth muscle in response to inflammatory cytokines . The dog is an important large animal used for cardiovascular research including effects of exercise, heart failure, and allograft rejection . However, molecular probes for iNOS developed in other mammals have not been reliable for the study of iNOS induction in canine vascular smooth muscle . Experiments were designed to develop a molecular probe for canine iNOS . Smooth muscle cells were isolated from canine aortas . The cells (passages 3-10) were incubated for 1, 3, 6, 12, 24, 48, or 72 h in the absence and presence of Escherichia coli lipopolysaccharide (LPS) to induce iNOS . Total RNA was isolated from the cells using standard techniques . RT-PCR with primers against conserved regions of all known iNOS enzyme was used to clone the iNOS cDNA . RT-PCR showed a single band only from cells treated with LPS . Cloned cDNA from cultured canine aortic smooth muscle cells has 84% homology to human, 81% to rat, and 81% to mouse iNOS gene . Identification of the cDNA for canine iNOS will be useful in the study of differential, transcriptional regulation of inducible (type II) compared with constitutive endothelial (type III) NOS in canine studies of allograft rejection and cardiovascular disease.

Curr Eye Res, 1998 Sep, 17(9), 941 - 6
The effect of genetic deficiency of adhesion molecules on the course of endotoxin-induced uveitis; Planck SR et al.; PURPOSE: Multiple adhesion molecules of the selectin, integrin, and immunoglobulin-like families are involved in the migration of leukocytes out of the bloodstream into inflamed tissues . This study addresses the question of which adhesion molecules are specifically involved in endotoxin-induced uveitis . METHODS: Mice genetically deficient in p-selectin, ICAM-1, beta2-integrin, or controls received intravitreal injections of endotoxin . Eyes were harvested 24 h later and inflammation was evaluated by histologic and immunohistochemical assays of infiltrating cells . RESULTS: Mice lacking either P-selectin or beta2-integrin had less inflammation than controls (median cells/section: 64 for P-selectin knockout vs 130 for controls, p=0.02, n=17 per group: 244 for beta2-integrin knockouts, n=14, vs 355 for controls, n=17, p=0.05) . Neither gene deletion significantly changed the ratio of infiltrating neutrophils to macrophages . ICAM-1 knockouts tended to have fewer infiltrating cells (median 22 cells/section) compared to controls (median 132 cells/section), but this difference was not statistically significant (p=0.25, n=9 per group) . CONCLUSIONS: P-selectin, beta2-integrin, and possibly ICAM-1 are involved in the ocular inflammatory response to endotoxin . The lack of complete inhibition of leukocyte infiltration with the complete loss of each adhesion molecule is in accord with the notion that alternative adhesion molecules also participate in this process.

Eur J Biochem, 1998 Aug 15, 256(1), 155 - 62
The catalytic mechanism of adenosylhomocysteine/methylthioadenosine nucleosidase from Escherichia coli--chemical evidence for a transition state with a substantial oxocarbenium character; Allart B et al.; The substrate and inhibitory specificity of Escherichia coli adenosylhomocysteine (AdoHcy)/methylthioadenosine (MeSAdo) nucleosidase has been explored with several MeSAdo analogues modified on the sugar moiety at the 2', 3' and 5' positions . Alteration at C3' or at C2' and C3' positions in MeSAdo abolished substrate activity . However, the 2'-deoxy analogue of MeSAdo is effective as a substrate; this result provides evidence against a possible general-base catalysis involving the anchimeric assistance of the 2'-alpha-hydroxy group and the formation of a 1,2-epoxide as an intermediate in the catalytic process . The results of a study of the interaction of an 8,5'-cyclo analogue of MeSAdo with the enzyme indicate the importance of the glycosidic conformation of the substrate for binding to the active site . The enzyme discriminates against methanol attack from the solvent during catalysis . This implies the participation of an enzyme-directed water nucleophile . A poor solvent kinetic deuterium-isotope effect was measured (0.93) on the Vmax . Plots of log Vmax and log (Vmax/Km) for MeSAdo as a function of pH values from 5.0 to 8.5 are similar, with two presumably essential ionisable groups for catalysis with apparent pKa values of 5.6 and 8.2, whereas Km is independent of pH . When the 2'-alpha-hydroxy group of MeSAdo is substituted by fluorine, a significant decrease (28 500-fold) in the Vmax for enzyme-catalysed hydrolysis of the modified substrate is observed . This result indicates a transition state with a substantial oxocarbenium character . From these data, the reaction mechanism for AdoHcy/MeSAdo nucleosidase is discussed.

Eur J Biochem, 1998 Aug 15, 256(1), 128 - 35
Interactions of non-detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation; Vuillard L et al.; Non-detergent sulfobetaines (NDSB) are a family of solubilizing and stabilizing agents for proteins . In a previous study {Goldberg, M . E., Expert-Bezancon, N., Vuillard, L . & Rabilloud, T . (1996) Folding & Design 1, 21-27} we showed that the amount of active protein recovered in in vitro folding experiments could be significantly increased by some NDSBS . In this work we investigated the mechanisms by which these molecules facilitate protein renaturation . Stopped-flow and manual-mixing fluorescence and enzyme activity measurements were used to compare the kinetics of protein folding in the presence and absence of N-phenyl-methyl-N,N-dimethylammonium-propane-sulfonate (NDSB 256) . Hen lysozyme and the beta2 subunit of Escherichia coli tryptophan synthase were chosen as model systems since their folding pathways had been previously investigated in detail . It is shown that, massive aggregation of tryptophan synthase occurs within less than 2.5 s after dilution in the renaturation buffer, but can be prevented by NDSB 256; only very early folding phases (such as the formation of a loosely packed hydrophobic core able to bind 8-anilino-1-naphthalenesulphonic acid, and the initial burying of tryptophan 177) are significantly altered by NDSB 256; none of the later phases is affected . Furthermore, NDSB 256 did not significantly affect any of the kinetic phases observed during the refolding of denatured lysozyme retaining intact disulphide bonds . This shows that NDSB 256 only interferes with very early steps in the folding process and acts by limiting the abortive interactions that could lead to the formation of inactive aggregates.

Mol Cell Biochem, 1998 Jul, 184(1-2), 311 - 20
Subtleties in control by metabolic channelling and enzyme organization; Kholodenko BN et al.; Because of its importance to cell function, the free-energy metabolism of the living cell is subtly and homeostatically controlled . Metabolic control analysis enables a quantitative determination of what controls the relevant fluxes . However, the original metabolic control analysis was developed for idealized metabolic systems, which were assumed to lack enzyme-enzyme association and direct metabolite transfer between enzymes (channelling) . We here review the recently developed molecular control analysis, which makes it possible to study non-ideal (channelled, organized) systems quantitatively in terms of what controls the fluxes, concentrations, and transit times . We show that in real, non-ideal pathways, the central control laws, such as the summation theorem for flux control, are richer than in ideal systems: the sum of the control of the enzymes participating in a non-ideal pathway may well exceed one (the number expected in the ideal pathways), but may also drop to values below one . Precise expressions indicate how total control is determined by non-ideal phenomena such as ternary complex formation (two enzymes, one metabolite), and enzyme sequestration . The bacterial phosphotransferase system (PTS), which catalyses the uptake and concomitant phosphorylation of glucose (and also regulates catabolite repression) is analyzed as an experimental example of a non-ideal pathway . Here, the phosphoryl group is channelled between enzymes, which could increase the sum of the enzyme control coefficients to two, whereas the formation of ternary complexes could decrease the sum of the enzyme control coefficients to below one . Experimental studies have recently confirmed this identification, as well as theoretically predicted values for the total control . Macromolecular crowding was shown to be a major candidate for the factor that modulates the non-ideal behaviour of the PTS pathway and the sum of the enzyme control coefficients.

Hum Mutat, 1998, 12(4), 267 - 73
Dihydropteridine reductase deficiency: physical structure of the QDPR gene, identification of two new mutations and genotype-phenotype correlations; Dianzani I et al.; Dihydropteridine reductase (DHPR) is an enzyme involved in recycling of tetrahydrobiopterin (BH4), the cofactor of the aromatic amino acid hydroxylases . Its deficiency is characterized by hyperphenylalaninemia due to the secondary defect of phenylalanine hydroxylase and depletion of the neurotransmitters dopamine and serotonin, whose syntheses are controlled by tryptophan and tyrosine hydroxylases . The DHPR cDNA has been cloned and mapped on 4p15.3 . In the present study we report the genomic structure of the DHPR gene (QDPR) . This gene includes seven exons within a range of 84-564 bp; the corresponding introns are flanked by canonic splice junctions . We also present a panel of PCR primers complementary to intronic sequences that greatly facilitates amplification of the gene and provides a genomic DNA approach for mutation detection . We have used this approach to study six patients with DHPR deficiency . Four known mutations (G23D, H158Y, IVS5G+ 1A, R221X) and two new mutations (Y150C and G218ins9bp) were found . The Y150C mutation was found in compound heterozygosity with G23D, a mutation always associated with a severe phenotype in homozygous patients . This patient has an intermediate phenotype (good response to monotherapy with BH4) . The mutant enzyme for Y150C was expressed in an E . coli system . Comparison of its kinetic parameters with those of the G23D mutant enzyme showed that it is not as effective as the wild-type enzyme, but is more active than the G23D mutant . This patient's intermediate phenotype is thus due to the mild DHPR mutation Y150C . Correlations between genotypes and phenotypes were also found for the other mutations.

Nature, 1998 Sep 10, 395(6698), 137 - 43
Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-gamma; Nolte RT et al.; The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-dependent transcription factor that is important in adipocyte differentiation and glucose homeostasis and which depends on interactions with co-activators, including steroid receptor co-activating factor-1 (SRC-1) . Here we present the X-ray crystal structure of the human apo-PPAR-gamma ligand-binding domain (LBD), at 2.2 A resolution; this structure reveals a large binding pocket, which may explain the diversity of ligands for PPAR-gamma . We also describe the ternary complex containing the PPAR-gamma LBD, the antidiabetic ligand rosiglitazone (BRL49653), and 88 amino acids of human SRC-1 at 2.3 A resolution . Glutamate and lysine residues that are highly conserved in LBDs of nuclear receptors form a 'charge clamp' that contacts backbone atoms of the LXXLL helices of SRC-1 . These results, together with the observation that two consecutive LXXLL motifs of SRC-1 make identical contacts with both subunits of a PPAR-gamma homodimer, suggest a general mechanism for the assembly of nuclear receptors with co-activators.

FEBS Lett, 1998 Aug 21, 433(3), 279 - 82
Pyridoxal phosphate binding to wild type, W330F, and C298S mutants of Escherichia coli apotryptophanase: unraveling the cold inactivation; Erez T et al.; The mechanism of pyridoxal phosphate (PLP) binding to apotryptophanase was investigated using stopped-flow kinetics with wild type (WT), W330F and C298S mutants . Based on the dependence of the rate constants on PLP concentrations for the fast and slow phases detected, two mechanistic schemes were proposed . For the WT and C298S mutant, the slow process is due to an isomerization of the aldimine complex after its formation, and not to the binding to an alternative conformation of the apoenzyme, which is the case proposed for the W330F mutant . It is suggested that during the cold inactivation process a conformational change precedes the aldimine bond cleavage . For the W330F apotryptophanase, another conformational change occurs subsequent to the aldimine bond cleavage.

FEBS Lett, 1998 Aug 21, 433(3), 205 - 10
Drosophila melanogaster acylphosphatase: a common ancestor for acylphosphatase isoenzymes of vertebrate species; Pieri A et al.; An open reading frame encoding a putative acylphosphatase was found in Drosophila melanogaster . The corresponding gene product shows 40% identity and 22 additional amino acid residues at the C-terminus as compared to muscle- and common-type human acylphosphatases . Moreover, all the residues involved in the catalytic mechanism of vertebrate enzymes are conserved in the D . melanogaster acylphosphatase . The D . melanogaster protein and a deletion mutant, similar in length to vertebrate acylphosphatases, were produced by cloning the corresponding cDNA in Escherichia coli . The wild-type enzyme is a protein with a well-established three-dimensional fold and a markedly reduced conformational stability as compared to vertebrate isoenzymes . The specific activity of the enzyme is significantly lower than that found in vertebrate enzymes though the substrate binding capability is basically unaltered . The deletion of 22 residues does not cause a significant change in k(cat), while affecting the apparent binding parameters . This work suggests that the genes encoding the vertebrate enzymes originate from an ancestor gene by duplication and subsequent evolution.

FEBS Lett, 1998 Aug 21, 433(3), 185 - 90
Cse1p functions as the nuclear export receptor for importin alpha in yeast; Kunzler M et al.; CSE1 is essential for yeast cell viability and has been implicated in chromosome segregation . Based on its sequence similarity, Cse1p has been grouped into the family of importin beta-like nucleocytoplasmic transport receptors with highest homology to the recently identified human nuclear export receptor for importin alpha, CAS . We demonstrate here that Cse1p physically interacts with yeast Ran and yeast importin alpha (Srp1p) in the yeast two-hybrid system and that recombinant Cse1p, Srp1p and Ran-GTP form a trimeric complex in vitro . Re-export of Srp1p from the nucleus into the cytoplasm and nuclear uptake of a reporter protein containing a classical NLS are inhibited in a cse1 mutant strain . These findings suggest that Cse1p is the exportin of importin alpha in yeast.

Carcinogenesis, 1998 Aug, 19(8), 1339 - 43
Differential cleavage of oligonucleotides containing the benzene-derived adduct, 1,N6-benzetheno-dA, by the major human AP endonuclease HAP1 and Escherichia coli exonuclease III and endonuclease IV; Hang B et al.; We report here that the newly synthesized DNA adduct, 1,N6-benzetheno-dA (pBQ-dA), in defined oligonucleotides {Chenna and Singer, Chem . Res . Toxicol., 8, 865-874}, is a substrate for the major human AP endonuclease, HAP1, and the Escherichia coli AP endonucleases, exonuclease III and endonuclease IV . The mechanism of cleavage is identical to that reported previously for 3,N4-benzetheno-dC (pBQ-dC) and leads to a phosphodiester bond cleavage 5' to the adduct . There are, however, significant differences in the rate of cleavage of this adduct by these enzymes . The two bacterial AP endonucleases are both much more efficient than the human repair enzyme . In addition, using two random oligodeoxynucleotide sequences containing a single pBQ-dA, exonuclease III and endonuclease IV are similarly active, while HAP1 shows a distinct sequence preference of approximately 10-fold in efficiency of cleavage . The repair of this adduct by the three recombinant enzymes is further confirmed by using both active site mutant HAP1 proteins and by E.coli mutant strains lacking exonuclease III and/ or endonuclease IV . This sequence-dependent repair of pBQ-dA by HAP1 may play an important role in modulating benzene-induced carcinogenesis.

Plant J, 1998 Jul, 15(1), 99 - 107
Phytobilin biosynthesis: cloning and expression of a gene encoding soluble ferredoxin-dependent heme oxygenase from Synechocystis sp . PCC 6803; Cornejo J et al.; The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IX alpha, in a reaction catalyzed by heme oxygenase . A gene containing an open reading frame with a predicted polypeptide that has a sequence similar to that of a conserved region of animal microsomal heme oxygenases was identified in the published genomic sequence of Synechocystis sp . PCC 6803 . This gene, named ho1, was cloned and expressed in Escherichia coli under the control of the lacZ promoter . Cells expressing the gene became green colored due to the accumulation of biliverdin IX alpha . The size of the expressed protein was equal to the predicted size of the Synechocystis gene product, named HO1 . Heme oxygenase activity was assayed in incubations containing extract of transformed E . coli cells . Incubations containing extract of induced cells, but not those containing extract of uninduced cells, had ferredoxin-dependent heme oxygenase activity . With mesoheme as the substrate, the reaction product was identified as mesobiliverdin IX alpha by spectrophotometry and reverse-phase HPLC . Heme oxygenase activity was not sedimented by centrifugation at 100, 000 g . Expression of HO1 increased several-fold during incubation of the cells for 72 h in iron-deficient medium.

Rev Argent Microbiol, 1998 Apr-Jun, 30(2), 64 - 72
{Obtaining antibodies and development of an immunoassay for the detection of Escherichia coli proteins in preparations of human recombinant gamma interferon}; Diaz N et al.; The present paper refers to the obtainment of polyspecific antisera directed against Escherichia coli host strain used to produce recombinant human gamma interferon (rec . hum . gamma IFN) . The antisera were obtained by the cascade immunization method . The animals (n = 3) were initially immunized with an E . coli protein preparation (EcPp) of the host strain obtained from a blank run which assures the absence of the rec . hum . gamma IFN protein . Afterwards, consecutive immunizations were carried out with the less immunogenic proteins . To obtain those proteins, EcPp is passed through a column containing antibodies purified from previous inoculations coupled to a gel matrix . In this way, the proteins that have not been recognized by the immune system in that moment (e.g . do not have their corresponding antibodies coupled to the column) are separated an used to reimmunize the animals . The analyses of the antisera by Western blotting show a progressive recognition of the host strain proteins by the antisera with the progression of the cascade method . The recognition is evident through all the molecular weight range . Those antisera were used as quality control of the recombinant protein, by quantification of the host strain protein contaminants using a multiantigenic ELISA . The detection limit of that system was 3.125 ng/mL and the quantification limit 6.25 ng/mL.

J Mol Biol, 1998 Oct 2, 282(4), 875 - 89
Structures of free and complexed forms of Escherichia coli xanthine-guanine phosphoribosyltransferase; Vos S et al.; Structures of free, substrate-bound and product-bound forms of Escherichia coli xanthine-guanine phosphoribosyltransferase (XGPRT) have been determined by X-ray crystallography . These are compared with the previously determined structure of magnesium and sulphate-bound XPRT . The structure of free XGPRT at 2.25 A resolution confirms the flexibility of residues in and around a mobile loop identified in other PRTases and shows that the cis-peptide conformation of Arg37 at the active site is maintained in the absence of bound ligands . The structures of XGPRT complexed with the purine base substrates guanine or xanthine in combination with cPRib-PP, an analog of the second substrate PRib-PP, have been solved to 2.0 A resolution . In these two structures the disordered phosphate-binding loop of uncomplexed XGPRT becomes ordered through interactions with the 5'-phosphate group of cPRib-PP . The cyclopentane ring of cPRib-PP has the C3 exo pucker conformation, stabilised by the cPRib-PP-bound Mg2+ . The purine base specificity of XGPRT appears to be due to water-mediated interactions between the 2-exocyclic groups of guanine or xanthine and side-chains of Glu136 and Asp140, as well as the main-chain oxygen atom of Ile135 . Asp92, together with Lys115, could help stabilise the N7-protonated tautomer of the incoming base and could act as a general base to remove the proton from N7 when the nucleotide product is formed . The 2.6 A resolution structure of XGPRT complexed with product GMP is similar to the substrate-bound complexes . However, the ribose ring of GMP is rotated by approximately 24 degrees compared with the equivalent ring in cPRib-PP . This rotation results in the loss of all interactions between the ribosyl group and the enzyme in the product complex .

J Mol Biol, 1998 Oct 2, 282(4), 775 - 87
Replication of R6K gamma origin in vitro: discrete start sites for DNA synthesis dependent on pi and its copy-up variants; Chen D et al.; The regulation of the plasmid R6K gamma origin (gamma ori) is accomplished through the ability of the pi protein to act as an initiator and inhibitor of replication . Hyperactive variants of this protein, called copy-up pi, allow four to tenfold increases of gamma ori plasmid DNA in vivo . The higher activity of copy-up pi variants could be explained by an increase in the initiator function, a decrease in the inhibitor activity, or a derepression of a more efficient mechanism of replication that can be used by wt pi (pi35 . 0) only under certain conditions . We have compared the replication activities of wt pi35.0 and copy-up pi mutants in vitro, and analyzed the replication products . It is shown that copy-up variants are several-fold more active than wt pi35.0 in replication . This appears to be due to enhanced specific replication activity of copy-up mutants rather than elevated fractions of protein proficient in DNA binding . Furthermore, biochemical complementation revealed that pi200 (copy-up) is dominant over wt pi35.0 . The elevated activity of copy-up pi is not caused by an increased rate of replisome assembly as inferred from in vitro replication assays in which the lag periods observed were similar to that of wt pi35.0 . Moreover, only one round of semiconservative, unidirectional replication occurred in all the samples analyzed indicating that copy-up pi proteins do not initiate multiple rounds of DNA synthesis . Rather, a larger fraction of DNA template replicates in the presence of copy-up pi as determined by electron microscopy . Two clusters of discrete DNA synthesis start sites are mapped by primer extension near the stability (stb) locus of the gamma ori . We show that the start sites are the same in the presence of wt pi35.0 or copy-up proteins . This comparative analysis suggests that wt pi35.0 and copy-up variants utilize fundamentally similar mechanism(s) of replication priming .

J Mol Biol, 1998 Oct 2, 282(4), 703 - 11
Functional analysis of the Escherichia coli genome using the sequence-to-structure-to-function paradigm: identification of proteins exhibiting the glutaredoxin/thioredoxin disulfide oxidoreductase activity; Fetrow JS et al.; The application of an automated method for the screening of protein activity based on the sequence-to-structure-to-function paradigm is presented for the complete Escherichia coli genome . First, the structure of the protein is identified from its sequence using a threading algorithm, which aligns the sequences to the best matching structure in a structural database and extends sequence analysis well beyond the limits of local sequence identity . Then, the active site is identified in the resulting sequence-to-structure alignment using a "fuzzy functional form" (FFF), a three-dimensional descriptor of the active site of a protein . Here, this sequence-to-structure-to-function concept is applied to analysis of the complete E . coli genome, i.e . all E . coli open reading frames (ORFs) are screened for the thiol-disulfide oxidoreductase activity of the glutaredoxin/thioredoxin protein family . We show that the method can identify the active sites in ten sequences that are known to or proposed to exhibit this activity . Furthermore, oxidoreductase activity is predicted in two other sequences that have not been identified previously . This method distinguishes protein pairs with similar active sites from proteins pairs that are just topological cousins, i.e . those having similar global folds, but not necessarily similar active sites . Thus, this method provides a novel approach for extraction of active site and functional information based on three-dimensional structures, rather than simple sequence analysis . Prediction of protein activity is fully automated and easily extendible to new functions . Finally, it is demonstrated here that the method can be applied to complete genome database analysis .

J Immunol, 1998 Sep 15, 161(6), 3019 - 25
Prostaglandin E2 protects against liver injury after Escherichia coli infection but hampers the resolution of the infection in mice; Takano M et al.; cAMP-increasing agents such as prostaglandin E2 (PGE2) are known to protect against LPS-induced liver injury by downregulating the production of inflammatory cytokines such as TNF-alpha . However, the effects of such reagents on host defense against bacterial infection remain unknown . We show here that in vivo administration of PGE2 significantly protected mice against liver injury after Escherichia coli infection but hampered the resolution of the infection . PGE2 significantly suppressed circulating TNF-alpha and IL-12 levels but increased the IL-10 production after E . coli challenge . PGE2 inhibited the emergence of gammadelta T cells in the peritoneal cavity, which are important for host defense against E . coli, and deteriorated bacterial exclusion in the peritoneal cavity after E . coli challenge . These results suggested that PGE2 affects host defense mechanisms against E . coli infection through modulation of cytokine production and gammadelta T cell accumulation.

FEBS Lett, 1998 Sep 4, 434(3), 401 - 5
The proregion of papaya proteinase IV inhibits Colorado potato beetle digestive cysteine proteinases; Visal S et al.; Three distinct digestive protease systems were induced in larvae of the herbivorous pest, Colorado potato beetle (CPB; Leptinotarsa decemlineata Say), and used as a model to assess the ability of the proregion of papaya proteinase IV (PPIV; glycyl endopeptidase, EC 3.4.22.25) to act as an inhibitor of insect digestive cysteine proteinases . As shown by gelatin/PAGE and complementary inhibition assays, a recombinant form of the proregion produced in Escherichia coli inhibited a fraction of the insect proteases also inhibited by the well-characterized inhibitor of cysteine proteinases, oryzacystatin I (OCI) . In contrast with OCI, the inhibitory potency of the proregion was affected by an increase of the temperature, suggesting a certain alteration of its structural integrity by the insect non-target proteases . This apparent susceptibility to proteolysis was confirmed by SDS-PAGE, after challenging the proregion with the different insect extracts . As seen on gel, selective inhibition of the insect aspartate proteinase, cathepsin D, with the inhibitor pepstatin A preserved the activity of the proregion against cysteine proteinases by preventing its hydrolysis . Taken together, these observations suggest the potential of plant protease proregions as regulators of cysteine proteinases in biotechnological systems, and show the ability of protease inhibitors to preserve the integrity of 'companion' defense-related proteins from the action of insensitive proteases in target pests.

FEBS Lett, 1998 Sep 4, 434(3), 372 - 6
In vitro heat effect on heterooligomeric subunit assembly of thermostable indolepyruvate ferredoxin oxidoreductase; Siddiqui MA et al.; Indolepyruvate ferredoxin oxidoreductase (IOR) from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 catalyzes the oxidative decarboxylation of arylpyruvates by forming a heterooligomeric complex (alpha2beta2) . The genes iorA and iorB which encode respective alpha and beta subunits, were coexpressed heterologously in Escherichia coli cells under anaerobic conditions . IOR activity was detected from the cell extract containing both subunits and its activity was enhanced by in vitro heat treatment prior to the assay . The iorA and iorB were expressed individually and each subunit was examined for enzymatic activity with and without heat treatment . IOR activity was detected neither from the extract of alpha subunit nor beta subunit . The alpha and beta subunits were mixed and then IOR activity was examined . Weak IOR activity was detected without heat treatment, however, upon heat treatment its activity was enhanced . The mixture of individually heat treated alpha and beta subunits did not possess any IOR activity even though the mixed sample was heat treated again . IOR alpha and beta subunits were individually purified to homogeneity, mixed with or without heat treatment and subunit assembly was examined by determining molecular mass . Upon heat treatment, inactive alpha and beta were converted to an active high molecular weight complex (195 kDa) which corresponds to the alpha2beta2 structure . However, the active complex was not formed without heat treatment, suggesting that high temperature environments are important for the heterooligomerization of IOR subunits.

FEBS Lett, 1998 Sep 4, 434(3), 289 - 94
A new human nm23 homologue (nm23-H5) specifically expressed in testis germinal cells; Munier A et al.; We have identified a cDNA encoding a 212 amino acid protein (Nm23-H5) with 27-31% identity to the human members of the nm23/nucleoside diphosphate (NDP) kinase gene family . The nm23-H5 gene is located on chromosome 5q23-31 and is transcribed as one main transcript of 1.1 kb which is highly and specifically expressed in testis, in the spermatogonia and early spermatocytes . Nm23-H5 possesses most of the residues conserved among NDP kinases plus an additional COOH-terminus end of 55 amino acids unique to this protein . However, under usual assay conditions, Nm23-H5 expressed in Escherichia coli did not exhibit NDP kinase activity . These results suggest that Nm23-H5 is specifically involved in early stages of spermatogenesis.

Curr Biol, 1998 Aug 27, 8(17), R619 - 21
Organelle division: from coli to chloroplasts; Lutkenhaus J; FtsZ, an ancestral homolog of eukaryotic tubulin, assembles into the cytokinetic Z ring that directs cell division in bacteria . Recent results indicate that FtsZ is also used for division by chloroplasts, though not by mitochondria.

Nucleic Acids Res, 1998 Oct 1, 26(19), 4524 - 8
Purification of plasmids by triplex affinity interaction; Schluep T et al.; Production of pharmaceutical grade plasmid DNA is an important issue in gene therapy . We developed a method for affinity purification of plasmids by triple helix interaction . This method is based on sequence-specific binding of an oligonucleotide immobilized on a large pore chromatography support to a target sequence on the plasmid . Using design criteria derived from thermodynamic data, we produced a 15mer target sequence which binds strongly to the affinity support under mildly acidic conditions . Plasmid DNA was purified from clarified Escherichia coli lysate by incubation with the affinity beads at pH 5.0 and high NaCl concentration . After extensive washing of the beads, purified plasmid DNA was eluted with alkaline buffer . The purified plasmid showed no RNA or cell DNA contamination in HPLC analysis and total protein concentration was reduced considerably . Due to its mechanical stability and porosity this support can be used in a continuous affinity purification process, which has a high potential for scale up.

Nucleic Acids Res, 1998 Oct 1, 26(19), 4446 - 53
Different cleavage specificities of RNases III from Rhodobacter capsulatus and Escherichia coli; Conrad C et al.; 23S rRNA in Rhodobacter capsulatus shows endoribonuclease III (RNase III)-dependent fragmentation in vivo at a unique extra stem-loop extending from position 1271 to 1331 . RNase III is a double strand (ds)-specific endoribonuclease . This substrate preference is mediated by a double-stranded RNA binding domain (dsRBD) within the protein . Although a certain degree of double strandedness is a prerequisite, the question arises what structural features exactly make this extra stem-loop an RNase III cleavage site, distinguishing it from the plethora of stem-loops in 23S rRNA? We used RNase III purified from R.capsulatus and Escherichia coli, respectively, together with well known substrates for E.coli RNase III and RNA substrates derived from the special cleavage site in R.capsulatus 23S rRNA to study the interaction between the Rhodobacter enzyme and the fragmentation site . Although both enzymes are very similar in their amino acid sequence, they exhibit significant differences in binding and cleavage of these in vitro substrates.

Nucleic Acids Res, 1998 Oct 1, 26(19), 4439 - 45
Analysis of the subunit assembly of the typeIC restriction-modification enzyme EcoR124I; Janscak P et al.; Type I restriction-modification (R-M) enzymes are composed of three different subunits, of which HsdS determines DNA specificity, HsdM is responsible for DNA methylation and HsdR is required for restriction . The HsdM and HsdS subunits can also form an independent DNA methyltransferase with a subunit stoichiometry of M2S1 . We found that the purified Eco R124I R-M enzyme was a mixture of two species as detected by the presence of two differently migrating specific DNA-protein complexes in a gel retardation assay . An analysis of protein subunits isolated from the complexes indicated that the larger species had a stoichiometry of R2M2S1and the smaller species had a stoichiometry of R1M2S1 . In vitro analysis of subunit assembly revealed that while binding of the first HsdR subunit to the M2S1complex was very tight, the second HsdR subunit was bound weakly and it dissociated from the R1M2S1complex with an apparent K d of approximately 2.4 x 10(-7) M . Functional assays have shown that only the R2M2S1complex is capable of DNA cleavage, however, the R1M2S1complex retains ATPase activity . The relevance of this situation is discussed in terms of the regulation of restriction activity in vivo upon conjugative transfer of a plasmid-born R-M system into an unmodified host cell.

Nucleic Acids Res, 1998 Oct 1, 26(19), 4409 - 12
Characterization of two intein homing endonucleases encoded in the DNA polymerase gene of Pyrococcus kodakaraensis strain KOD1; Nishioka M et al.; Two intein endonucleases, denoted PI- Pko I and PI- Pko II, in the DNA polymerase gene of the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 were expressed in Escherichia coli and the recombinant endonucleases were characterized . Both endonucleases were thermostable and cleaved their intein-less DNA sequences leaving four base 3'-hydroxyl overhangs . PI-Pko I exhibited 22 times higher specific activity than PI-Pko II and the activity of PI-Pko II was enhanced at higher potassium ion concentrations (1 M) . Recognition sequences were also determined using synthetic oligonucleotides inserted into plasmid pUC19 . It was shown that DNA sequences of 19 and 16 bp are needed for cleavage by PI-Pko I and PI-Pko II, respectively . PI-Pko II could cleave the downstream junction region between intein-encoding and mature DNA polymerase regions and cleavage by PI-Pko II could be detected even when chromosomal DNA of P.kodakaraensis KOD1 was used as substrate . Therefore, it is suggested that these endonucleases are switching endonucleases whose function lies in the rearrangement of chromosomal DNA.

Nucleic Acids Res, 1998 Oct 1, 26(19), 4347 - 55
DNA complexes obtained with the integron integrase IntI1 at the attI1 site; Gravel A et al.; Integrons are genetic elements that are able to capture genes by a site-specific recombination mechanism . Integrons contain a gene coding for a lambda-like integrase that carries out site-specific recombination by interacting with two different target sites; the attI site and the palindromic sequence attC (59 base element) . Cassette integrations usually involve the attI site, while cassette excisions use attC . Therefore, the integrase should bind both sites to cleave DNA and perform site-specific recombination reactions . We have used purified maltose-binding protein fused with the integrase (MBP-IntI1) and native IntI1 protein and gel retardation assays with fragments containing the complete and partial attI1 site to show formation of four complexes in this region . Chemical modification of specific nucleotides within the attI1 site was used to investigate their interference with binding of the integrase protein . We attribute IntI1 specific binding to four regions in the attI1 site and a GTTA consensus sequence is found in three of the four regions . Interference by modified guanine and thymine residues in the DNA major groove and adenine residues in the minor groove were observed, indicating that the integrase interacts with both sides of the helix . Binding of IntI1 to attC is also discussed.

Nucleic Acids Res, 1998 Oct 1, 26(19), 4339 - 46
In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/translation; Ohuchi S et al.; A novel in vitro method for the generation of a protein library has been developed using the polymerase chain reaction (PCR) amplification of a single DNA molecule followed by in vitro coupled transcription/translation . DNA template encoding green fluorescent protein of a jellyfish Aequorea victoria was extensively diluted to one molecule per well, and then amplified by a total of 80 cycles of PCR with nested primers . The exact number of origins in the amplified DNA fragment was then estimated by directly sequencing a part of the fragment, at which an individual template molecule was marked by PCR with a primer containing three randomized bases . Since the sequences obtained in 91 independent amplifications were diversified statistically, each amplified fragment was likely originated from a single DNA molecule . In addition, the amplified fragments served as a template for in vitro coupled transcription/translation using T7 RNA polymerase and Escherichia coli S30 extract . These results suggest that the library obtained by the PCR amplification of a single DNA molecule diluted from a variety of DNA pools is potentially useful in high-throughput generation of protein libraries.

Proteins, 1998 Oct 1, 33(1), 135 - 43
Folding the ribonuclease H domain of Moloney murine leukemia virus reverse transcriptase requires metal binding or a short N-terminal extension; Goedken ER et al.; Reverse transcriptase (RT) is a modular enzyme carrying polymerase and ribonuclease H (RNase H) activities in separable domains . Retroviral replication requires both of these activities . The RNase H domain is responsible for hydrolysis of the RNA portion of RNA x DNA hybrids, and this activity requires the presence of divalent cations (Mg2+ or Mn2+) that bind its active site . This domain is a part of a large family of homologous RNase H enzymes of which the RNase HI protein from Escherichia coli is the best characterized . Although the isolated RNase H domain from human immunodeficiency virus RT is inactive, the Moloney murine leukemia virus (MMLV) domain is active in the absence of the polymerase domain, making functional studies more accessible . Using circular dichroism spectroscopy, we characterized the stability and folding of two different fragments of MMLV RT that retain RNase H activity . The smaller fragment corresponding to the 157 C-terminal residues of RT is predominantly unfolded in the absence of divalent cations, but folding can be induced by the addition of metal . The larger fragment corresponding to the 175 C-terminal residues, however, is stably folded in the absence of metal . Thus, an 18 residue N-terminal extension outside the region homologous to E . coli RNase HI is important for the structural stability of the RNase H domain of MMLV RT . Therefore, this region should be considered part of the RNase H domain.

Mol Cell Biol, 1998 Oct, 18(10), 6063 - 74
FGF-18, a novel member of the fibroblast growth factor family, stimulates hepatic and intestinal proliferation; Hu MC et al.; The fibroblast growth factors (FGFs) play key roles in controlling tissue growth, morphogenesis, and repair in animals . We have cloned a novel member of the FGF family, designated FGF-18, that is expressed primarily in the lungs and kidneys and at lower levels in the heart, testes, spleen, skeletal muscle, and brain . Sequence comparison indicates that FGF-18 is highly conserved between humans and mice and is most homologous to FGF-8 among the FGF family members . FGF-18 has a typical signal sequence and was glycosylated and secreted when it was transfected into 293-EBNA cells . Recombinant murine FGF-18 protein (rMuFGF-18) stimulated proliferation in the fibroblast cell line NIH 3T3 in vitro in a heparan sulfate-dependent manner . To examine its biological activity in vivo, rMuFGF-18 was injected into normal mice and ectopically overexpressed in transgenic mice by using a liver-specific promoter . Injection of rMuFGF-18 induced proliferation in a wide variety of tissues, including tissues of both epithelial and mesenchymal origin . The two tissues which appeared to be the primary targets of FGF-18 were the liver and small intestine, both of which exhibited histologic evidence of proliferation and showed significant gains in organ weight following 7 (sometimes 3) days of FGF-18 treatment . Transgenic mice that overexpressed FGF-18 in the liver also exhibited an increase in liver weight and hepatocellular proliferation . These results suggest that FGF-18 is a pleiotropic growth factor that stimulates proliferation in a number of tissues, most notably the liver and small intestine.

J Histochem Cytochem, 1998 Oct, 46(10), 1103 - 11
Nuclear detection of cellular retinoic acid binding proteins I and II with new antibodies; Gaub MP et al.; Apart from the retinoic acid nuclear receptor family, there are two low molecular weight (15 kD) cellular retinoic acid binding proteins, named CRABPI and II . Mouse monoclonal and rabbit polyclonal antibodies were raised against these proteins by using as antigens either synthetic peptides corresponding to amino acid sequences unique to CRABPI or CRABPII, or purified CRABP proteins expressed in E . coli . Antibodies specific for mouse and/or human CRABPI and CRABPII were obtained and characterized by immunocytochemistry and immunoblotting . They allowed the detection not only of CRABPI but also of CRABPII in both nuclear and cytosolic extracts from transfected COS-1 cells, mouse embryos, and various cell lines.

J Lipid Res, 1998 Sep, 39(9), 1862 - 9
Baculovirus-mediated expression of recombinant rat phosphatidylcholine transfer protein; Feng L et al.; Phosphatidylcholine transfer protein catalyzes intermembrane transfer of phosphatidylcholines exclusive of all other phospholipid classes . Although postulated to participate in phosphatidylcholine biosynthesis and biliary trafficking in liver, the molecular basis underlying the substrate specificity of phosphatidylcholine transfer protein remains to be elucidated . Having demonstrated the inability of Escherichia coli to express recombinant phosphatidylcholine transfer protein, we infected Spodoptera frugiperda (Sf9) cells with recombinant baculovirus . When assayed in vitro, cytosol of recombinant but not control infected cells demonstrated high levels of intermembrane phosphatidylcholine transfer activity and no transfer activity for phosphatidylethanolamine . A two-step purification protocol in which 10 mg of cytosolic protein was subjected to anion exchange chromatography followed by hydroxylapatite chromatography yielded 0.1 mg active protein which was >92% pure . The identity of purified protein was confirmed by matrix-assisted laser desorption-ionization mass spectrometry and by amino acid sequencing . Based on the recovery of 30% of PC transfer activity after purification, we estimate that recombinant rat phosphatidylcholine transfer protein accounted for approximately 3-6% of cytosolic protein mass of infected cells . These results demonstrate the utility of baculovirus for expressing recombinant phosphatidylcholine transfer protein and should facilitate studies designed to elucidate the structural biology and physiological functions of this uniquely specific phospholipid transfer protein.

J Chromatogr A, 1998 Aug 7, 816(1), 107 - 11
Recent developments of magnetic beads for use in nucleic acid purification; Levison PR et al.; The performance of Magarose, an agarose-based bead containing a paramagnetic component has been evaluated . The anion exchanger DEAE-Magarose is effective at binding DNA from a crude cell lysate . The plasmid pBluescript was isolated from 1.5 ml Escherichia coli JM109 cell culture, following alkaline lysis yielding 8.2 micrograms high-quality DNA . Under similar binding conditions 21 micrograms of salmon sperm DNA bound to the ion exchangers . The affinity medium oligo-dT Magarose was demonstrated to bind 75 mumol of an oligo-dA probe/g of medium by hybridization . Under similar conditions mRNA could be isolated from a preparation of baby hamster cell total RNA . The magnetic susceptibility of Magarose is very high, facilitating the use of this separation technique for rapid batch chromatographic processes.

FEMS Microbiol Lett, 1998 Sep 1, 166(1), 121 - 6
Identification of a gene sequence encoding a putative pyruvate oxidoreductase in Serpulina pilosicoli; Rayment SJ et al.; Serpulina pilosicoli is a recently described species of intestinal spirochaete which can be identified using a species-specific monoclonal antibody BJL/AC1 reactive with a 29-kDa protein located in the cell envelope . A genomic library of the type strain of S . pilosicoli P43/6/78T was created in lambda zap express and screened using BJL/AC1 . Single positive clones were isolated and excised into the phagemid vector pBK-CMV . Phagemid DNA was purified and a single clone was selected for sequencing . The size of spirochaetal DNA insert was determined by digestion with restriction endonucleases EcoRI and PstI as being approximately 2.6 kb . The nucleotide sequence of the gene encoding the protein with which the antibody reacted was determined by cycle sequencing . The insert contained an open reading frame of 285 nucleotides . Translation of the nucleotide sequence into amino acid (aa) residues showed a sequence of 275 aa . Comparison of this sequence with databases revealed homology to pyruvate oxidoreductases from various organisms found in the gastroinestinal tract . These included the pyruvate ferredoxin oxidoreductase (POR) alpha submit of Helicobacter pylori (38.8% identity in 250 aa), pyruvate-flavodoxin oxidoreductase of Escherichia coli (28.7% identify in 258 aa) and Giardia intestinalis (25.1% identity in 251 aa) . A significant level of homology was also observed with hyperthermophilic bacteria such as the POR of Thermatoga maritima (38.6% in 254 aa) and the 2-ketovalerate-ferredoxin oxidoreductase of Pyrococcus furiosus (34% in 262 aa).

FEMS Microbiol Lett, 1998 Sep 1, 166(1), 71 - 8
A novel proline-rich protein, EspF, is secreted from enteropathogenic Escherichia coli via the type III export pathway; McNamara BP et al.; Enteropathogenic Escherichia coli (EPEC) cause a characteristic attaching and effacing (A/E) lesion in intestinal epithelial cells that is associated with the expression and export of specific bacterial proteins via a type III secretion pathway . These effector proteins and components of the type III export apparatus are encoded on a pathogenicity island known as the locus of enterocyte effacement (LEE) . In this study, we describe a proline-rich protein, EspF, encoded by the LEE that is secreted by the EPEC type III secretion apparatus . Whereas an espF deletion mutant does not synthesize or secrete EspF, surprisingly it retains the ability to induce host signaling events, perform A/E activities, and invade host epithelial cells . Although these results do not indicate on obvious role for EspF in the formation of A/E lesions nor in the invasion of epithelial cells, they do not preclude a role played by EspF in other aspects of EPEC pathogenesis.

RNA, 1998 Sep, 4(9), 1047 - 54
Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo; Perriman R et al.; Many proteins with unusual structural properties are comprised of multiple repeating amino acid sequences and are often fractious to expression in recombinant systems . To facilitate recombinant production of such proteins for structural and engineering studies, we have produced circular messenger RNAs with infinite open reading frames . We show that a circular mRNA containing a simple green fluorescent protein (GFP) open reading frame can direct GFP expression in Escherichia coli . A circular mRNA with an infinite GFP open reading frame produces extremely long protein chains, proving that bacterial ribosomes can internally initiate and repeatedly transit a circular mRNA . Only the monomeric forms of GFP produced from circular mRNA are fluorescent . Analysis of the translation initiation region shows that multiple sequences contribute to maximal translation from circular mRNA . This technology provides a unique means of producing a very long repeating-sequence protein, and may open the way for development of proteinaceous materials with novel properties.

Biochim Biophys Acta, 1998 Sep 16, 1404(3), 457 - 74
A monoclonal antibody to a multiphosphorylated, conformational epitope at the carboxy-terminus of p53; Otvos L Jr et al.; Mutations of the gene encoding the tumor suppressor protein p53 are the most common molecular alterations of cancer cells found in about half of all human tumors . Mutations which cluster in well-defined hot spots change the structure of the protein thus affecting its ability to bind to DNA . Post-translational modifications, primarily phosphorylation, might also influence how p53 binds to DNA or folds to its active tetrameric form . However, the lack of appropriate biochemical markers to characterize the status of phosphorylation in different cell types and in cells at different stages of tumor progression has prohibited such investigations . To generate a sensitive and phosphorylation-specific monoclonal antibody (mAb), we chemically synthesized the C-terminal 23 amino acid stretch of human p53 in a double-phosphorylated form . The peptide 371-393, carrying phosphate groups on Ser378 and Ser392, was co-synthesized with a turn-inducing spacer and peptide 31D, an immunodominant T-helper cell epitope in mice of the H-2k haplotype . After immunization and fusion of splenocytes with myeloma cells, a number of mAbs were obtained, from which mAb p53-18 emerged as a highly sensitive reagent . By enzyme-linked immunosorbent assay, p53-18, a mAb of the IgM isotype, recognized phosphorylated p53, expressed in insect cells infected with a recombinant baculovirus but not p53 expressed in Escherichia coli . Moreover, murine p53 from insect cells could be immune purified with mAb p53-18 . Mass spectrometry following tryptic digestion of the purified protein and liquid chromatography of the fragments verified the presence of phosphate groups at both Ser375 and Ser389 . From the corresponding human protein fragments, mAb p53-18 bound to the immunizing peptide phosphorylated on Ser378 and on Ser392, but failed to cross-react with the unphosphorylated peptide, or peptides phosphorylated individually on either Ser378 or Ser392 . The binding to the unphosphorylated peptide could be restored, however, if the peptide conformation was stabilized to that of an alpha-helix . The immunogenic nature of the multiphosphorylated C-terminus of p53 is indicated by the finding that human sera, mostly from cancer patients, preferentially recognized the double-phosphorylated peptide over the monophosphorylated or unphosphorylated analogs . Antibody p53-18 appears to be a highly useful biochemical marker to detect low levels of p53 protein in different tissues, and to be a key tool to characterize the phosphorylation status of the C-terminus of p53 protein originated from various sources.

Protein Eng, 1998 Jul, 11(7), 601 - 7
Improved folding of apo-retinol-binding protein in the periplasm of Escherichia coli: positive influences of dsbC coexpression and of an amino acid exchange in the vitamin A binding site; Schmidt AM et al.; The in vivo folding of the serum retinol-binding protein (RBP), a representative of the lipocalin structural family, is known to be complex . In order to gain insight into the essential steps along its folding pathway the heterologous production of the functional protein in Escherichia coli was investigated . Simultaneous overexpression of the bacterial dsbC gene, which codes for a periplasmic thiol-disulphide oxidoreductase, prevented the formation of soluble RBP variants with non-native disulphide bonds that were otherwise observed . Although the coexpression of dsbC had furthermore a stabilizing effect on the cell viability, the relative yield of the solubly produced RBP was not much better . In an attempt to enhance its folding efficiency, a favourable point mutation in the inner part of the retinol-binding pocket was predicted . Replacement of the polar Gln117 with an lie side chain seemed not only to relieve the unfavourable energetics of the carboxamide group in the environment of predominantly non-polar residues but also to fill an adjacent cavity in the hydrophobic core . Indeed, this single substitution reproducibly resulted in a more than threefold increase in the amount of functional recombinant RBP . Ligand binding experiments showed that the affinity of this mutant for retinol was slightly enhanced . Kinetic measurements revealed that this was due to a higher association rate whereas the dissociation of the complex with retinol was essentially unaffected . Although the question remained why nature did not select this obviously beneficial mutation, our results demonstrate that the folding pathway of a lipocalin can be optimized by protein engineering.

Protein Eng, 1998 Jul, 11(7), 583 - 8
Correct assembly of human normal adult hemoglobin when expressed in transgenic swine: chemical, conformational and functional equivalence with the human-derived protein; Manjula BN et al.; Structural and functional investigations of recombinant human hemoglobin A (HbA) isolated from the erythrocytes of transgenic swine coexpressing human alpha- and beta-globins have been carried out to authenticate its correct expression, post-translational processing and assembly . The HbA expressed in transgenic swine (TgHbA) is indistinguishable from the human-derived HbA in terms of its isoelectric pH, mass and elution pattern on a Mono S column . The chemical identity of the alpha- and beta-globin chains of TgHbA with the corresponding chains from human-derived HbA has been established by tryptic peptide mapping and amino acid sequencing . The proton NMR spectra of TgHbA have demonstrated that the conformational aspects of the protein around the heme pocket are indistinguishable from those of the control sample of HbA . The equivalence of the hydrogen bond pattern of TgHbA (in particular the inter-subunit surfaces) with that of authentic HbA has also been established by NMR studies . Consistent with these structural and conformational analyses, the TgHbA also exhibits complete functional equivalence with the human-derived HbA with respect to oxygen affinity, cooperativity, Bohr effect and allostery . Hence the studies presented here demonstrate that the transgenic swine system correctly transcribes the alpha- and beta-globin transgenes, translates the respective alpha- and beta-globin mRNA to generate the corresponding globin chains, carries out the correct cotranslational processing of the translated globin chains, inserts the heme into the globin chains in the same orientation as in the human-derived HbA and assembles the alpha- and beta-subunits into a functionally cooperative tetramer that exhibits a response to allosteric effectors identical with that of human-derived HbA . Thus, in the transgenic swine system, in vitro chemical manipulation steps such as those needed in the Escherichia coli and the yeast systems, to convert the rHbA expressed in these systems into forms functionally identical with that of the human-derived protein, are not needed . An additional advantage of the transgenic swine system is the stability of the transgenes over many generations . Hence the transgenic swine could serve as an excellent system for the production of human HbA (or its variants) for structure-function studies and for therapeutic applications.

Protein Eng, 1998 Jul, 11(7), 563 - 8
Mutation of cis-proline 207 in mitochondrial creatine kinase to alanine leads to increased acid stability; Forstner M et al.; We show that the mutation of an uncharged residue far from the active site to another uncharged residue can have effects on the active site without disturbing the overall structure of the protein . Cis-proline 207 of mitochondrial creatine kinase was mutated to alanine . The mutant showed a decrease in the pH-optimum for ATP synthesis by 1.5 units while the maximum relative activity was lowered to 53% of the wild-type enzyme . In the direction of ATP consumption, the pH optimum was lowered by 1.3 units and the maximum relative activity was 49% of the wild-type enzyme . The enzyme kinetic parameters Km and Kd for the substrates did not change dramatically, indicating a largely unperturbed active site . Small-angle X-ray scattering was used to investigate the structural change concomitant with the mutation, yielding a scattering profile only slightly different from that of the wild-type enzyme . Neither the radius of gyration nor the molecular mass showed any significant differences, leading to the conclusion that quarternary organization and fold of the mutant and the wild-type enzymes were similar . Theoretical analysis suggests the most probable primary source of structural change to be a transition of residue 207 peptide bond torsional angle co from the cis to the trans configuration.

Protein Eng, 1998 Jul, 11(7), 549 - 55
Truncation and heme pocket mutations reduce production of functional catalase HPII in Escherichia coli; Sevinc MS et al.; The subunit of catalase HPII from Escherichia coli is 753 residues in length and contains a core of approximately 500 residues, with high structural similarity to all other heme catalases . To this core are added extensions of approximately 80 and 180 residues at the N- and C-termini, respectively . The tetrameric structure is made up of a pair of interwoven dimers in which 90 N-terminal residues of each subunit are inserted through a loop formed by the hinge region linking the beta-barrel and alpha-helical domains of the adjacent subunit . A high concentration of proline residues is found in the vicinity of the overlap regions . To study the influence of the extended regions on folding and subunit association of HPII, a diversity of modifications have been introduced . Removal of the complete C-terminal domain or the N-terminal extension, either separately or together, effectively creating a small subunit catalase, resulted in no enzyme accumulation . Systematic truncations showed that only nine C-terminal residues (Ile745 to Ala753) could be removed without significantly affecting the accumulation of active enzyme . Removal or even conservative replacements of the side chain of Arg744 significantly reduced the accumulation of active enzyme despite this residue interacting only with the C-terminal domain . Removal of as few as 18 residues from the N-terminus also reduced accumulation of active enzyme . Changes to other residues in the protein, including residues in the heme binding pocket, also reduced the accumulation of active protein without substantially affecting the enzyme specific activity . Implications of these data for the interdependence of subunit folding and subunit-subunit interactions are discussed.

J Invest Dermatol, 1998 Sep, 111(3), 406 - 9
Mutations in the ferrochelatase gene of four Spanish patients with erythropoietic protoporphyria; Gouya L et al.; Erythropoietic protoporphyria is a hereditary disorder of porphyrin metabolism caused by mutations in the ferrochelatase gene . Ferrochelatase catalyzes the chelation of ferrous iron into protoporphyrin IX to form heme . Mutation analysis was performed in four Spanish erythropoietic protoporphyria families resulting in the identification of four different mutations in the ferrochelatase gene . Two of them were novel mutations, a missense mutation (1157 A-->C, H386P) and a frameshift mutation (843delC) found in two Spanish families, respectively . The third and the forth Spanish patients carried already published ferrochelatase gene mutations, a nonsense mutation (343C-->T, R115X) and a missense mutation (557T-->C, I186T), respectively . The newly described frameshift mutation (843delC) predicted formation of an abrupt mRNA . The deleterious effect of His386 to Pro substitution as a result of mutation 1157 A-->C on the ferrochelatase activity was investigated by expressing the mutant ferrochelatase in Escherichia coli . The mutant ferrochelatase exhibited only 0.8% of the wild-type ferrochelatase activity . Prediction of the secondary structure of ferrochelatase suggested that the H386P mutation disrupted the original alpha-helical structure by way of introducing a turn, a rather drastic structural change of the enzyme sufficient to cause activity loss.

RNA, 1998 Sep, 4(9), 1154 - 64
Structure of 5S rRNA within the Escherichia coli ribosome: iodine-induced cleavage patterns of phosphorothioate derivatives; Shpanchenko OV et al.; The protection patterns of 5S rRNA in solution, within the ribosomal 50S subunit, 70S ribosomes, and functional complexes, were assessed with the phosphorothioate method . About 20% of the analyzed positions (G9-G107) showed strong assembly defects: A phosphorothioate at one of these positions significantly impaired the incorporation of 5S rRNA into 50S particles . The reverse has also been observed: A phosphorothioate is preferred over a phosphate residue in the assembly process at a few positions . The results further demonstrate that 5S rRNA undergoes conformational changes during the assembly in the central protuberance of the 50S subunit and upon association with the small ribosomal subunit forming a 70S ribosome . In striking contrast, when the 70S ribosomes are once formed, the contact pattern of the 5S rRNA is the same in various functional states such as initiation-like complexes and pre- and posttranslocational states.

RNA, 1998 Sep, 4(9), 1134 - 53
The 5S rRNA loop E: chemical probing and phylogenetic data versus crystal structure; Leontis NB et al.; A significant fraction of the bases in a folded, structured RNA molecule participate in noncanonical base pairing interactions, often in the context of internal loops or multi-helix junction loops . The appearance of each new high-resolution RNA structure provides welcome data to guide efforts to understand and predict RNA 3D structure, especially when the RNA in question is a functionally conserved molecule . The recent publication of the crystal structure of the "Loop E" region of bacterial 5S ribosomal RNA is such an event {Correll CC, Freeborn B, Moore PB, Steitz TA, 1997, Cell 91:705-712} . In addition to providing more examples of already established noncanonical base pairs, such as purine-purine sheared pairings, trans-Hoogsteen UA, and GU wobble pairs, the structure provides the first high-resolution views of two new purine-purine pairings and a new GU pairing . The goal of the present analysis is to expand the capabilities of both chemical probing and phylogenetic analysis to predict with greater accuracy the structures of RNA molecules . First, in light of existing chemical probing data, we investigate what lessons could be learned regarding the interpretation of this widely used method of RNA structure probing . Then we analyze the 3D structure with reference to molecular phylogeny data (assuming conservation of function) to discover what alternative base pairings are geometrically compatible with the structure . The comparisons between previous modeling efforts and crystal structures show that the intricate involvements of ions and water molecules in the maintenance of non-Watson-Crick pairs render the process of correctly identifying the interacting sites in such pairs treacherous, except in cases of trans-Hoogsteen A/U or sheared A/G pairs for the adenine N1 site . The phylogenetic analysis identifies A/A, A/C, A/U and C/A, C/C, and C/U pairings isosteric with sheared A/G, as well as A/A and A/C pairings isosteric with both G/U and G/G bifurcated pairings . Thus, each non-Watson-Crick pair could be characterized by a phylogenetic signature of variations between isosteric-like pairings . In addition to the conservative changes, which form a dictionary of pairings isosterically compatible with those observed in the crystal structure, concerted changes involving several base pairs also occur . The latter covariations may indicate transitions between related but distinctive motifs within the loop E of 5S ribosomal RNA.

RNA, 1998 Sep, 4(9), 1111 - 23
Distinct functions of the closely related tandem RNA-recognition motifs of hnRNP A1; Mayeda A et al.; hnRNP A1 regulates alternative splicing by antagonizing SR proteins . It consists of two closely related, tandem RNA-recognition motifs (RRMs), followed by a glycine-rich domain . Analysis of variant proteins with duplications, deletions, or swaps of the RRMs showed that although both RRMs are required for alternative splicing function, each RRM plays distinct roles, and their relative position is important . Surprisingly, RRM2 but not RRM1 could support this function when duplicated, despite their very similar structure . Specific RNA binding and annealing are not sufficient for hnRNP A1 alternative splicing function . These observations, together with phylogenetic and structural data, suggest that the two RRMs are quasi-symmetric but functionally nonequivalent modules that evolved as components of a single bipartite domain.

Electrophoresis, 1998 Aug, 19(11), 2010 - 3
Charge heterogeneity of macrophage migration inhibitory factor (MIF) in human liver and breast tissue; Magi B et al.; Macrophage migration inhibitory factor (MIF) is an ubiquitous protein playing various immunological and hormonal roles . Theoretical electrophoretic coordinates calculated from protein sequence in the SWISS-PROT database (AC P14174) are 12 kDa and pI 8.24 . Using two-dimensional (2-D) immunoblotting, we have detected isoelectric forms at ca . 11.9 kDa, with pI values of 7.8 and 6.98 in human liver tissue, breast tissue and a cell line and in preparations of human MIF expressed in E . coli . This evidence suggests that MIF charge heterogeneity originates from a post-translational modification not requiring eukaryote-specific enzymes . We have also detected in human liver a minor immunoreactive spot at pI 6.23, which coincides with the MIF spot in the liver map in SWISS-2DPAGE . The pI 6.23 isoform also conceivably derives from post-translational modification, as MIF is known to be encoded in the human genome by a single copy gene.

Electrophoresis, 1998 Aug, 19(11), 1960 - 71
'98 Escherichia coli SWISS-2DPAGE database update; Tonella L et al.; The combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), computer image analysis and several protein identification techniques allowed the Escherichia coli SWISS-2DPAGE database to be established . This is part of the ExPASy molecular biology server accessible through the WWW at the URL address . Here we report recent progress in the development of the E . coli SWISS-2DPAGE database . Proteins were separated with immobilized pH gradients in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension . To increase the resolution of the separation and thus the number of identified proteins, a variety of wide and narrow range immobilized pH gradients were used in the first dimension . Micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and spots were visualized by amido black staining . Protein identification techniques such as amino acid composition analysis, gel comparison and microsequencing were used, as well as a recently described Edman "sequence tag" approach . Some of the above identification techniques were coupled with database searching tools . Currently 231 polypeptides are identified on the E . coli SWISS-2DPAGE map: 64 have been identified by N-terminal microsequencing, 39 by amino acid composition, and 82 by sequence tag . Of 153 proteins putatively identified by gel comparison, 65 have been confirmed . Many proteins have been identified using more than one technique . Faster progress in the E . coli proteome project will now be possible with advances in biochemical methodology and with the completion of the entire E . coli genome.

Electrophoresis, 1998 Aug, 19(11), 1941 - 9
Towards an automated approach for protein identification in proteome projects; Traini M et al.; The development of automated, high throughput technologies for the rapid identification of proteins is essential for large-scale proteome projects . While a degree of automation already exists in some stages of the protein identification process, such as automated acquisition of matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectra, efficient interfaces between different stages are still lacking . We report the development of a highly automated, integrated system for large scale identification of proteins separated by two-dimensional gel electrophoresis (2-DE), based on peptide mass fingerprinting . A prototype robotic system was used to image and excise 288 protein spots from an amido black stained polyvinylidene difluoride (PVDF) blot . Protein samples were enzymatically digested with a commercial automated liquid handling system . MALDI-TOF mass spectrometry was used to acquire mass spectra automatically, and the data analysed with novel automated peptide mass fingerprinting database interrogation software . Using this highly automated system, we were able to identify 95 proteins on the basis of peptide mass fingerprinting, isoelectric point and molecular weight, in a period of less than ten working days . Advantages, problems, and future developments in robotic excision systems, liquid handling, and automated database interrogation software are discussed.

Mutat Res, 1998 Aug 7, 408(2), 147 - 57
The use of oriC-dependent phage infection to characterize the ultra violet (UV)-induced inhibition of initiation of DNA replication in Escherichia coli; Coates NJ et al.; The oriC transducing phage lambda poriCc is a pseudovirulent phage capable of forming plaques on a lambda lysogen . This phenotype is dependent upon the presence of the oriC insert . The ability of lambda poriCc to form plaques on a lambda lysogen represents a potential phage assay system for studying aspects of oriC function . In the present study we establish that lambda poriCc infection of a lambda lysogen is a legitimate assay for oriC function . We use this assay to confirm the previously reported observation that initiation of DNA replication from oriC is transiently inhibited in a ultra violet (UV) irradiated cell at doses greater than 60 J/m2 . We further demonstrate using this assay that the UV induced inhibition of initiation of DNA replication from oriC is not a SOS function nor a heat shock function . In the course of these studies, we found that lambda poriCc infection of a non-lysogenic cell is extremely sensitive to pre-irradiation of the Escherichia coli host . We postulate that the sensitivity of lambda poriCc replication to host cell pre-irradiation reflects in some way the transient inhibition of initiation of DNA replication from oriC following UV irradiation.

Mutat Res, 1998 Aug 7, 408(2), 129 - 35
Mutational potentiality of stannous chloride: an important reducing agent in the Tc-99m-radiopharmaceuticals; Cabral RE et al.; Stannous chloride (SnCl2) is frequently used in nuclear medicine as a reducing agent to label many radiopharmaceutical products with technetium-99m (99mTc) . The aim of the present paper was to study the role of DNA repair genes in the repair of SnCl2-induced damage, using mutant strains of Escherichia coli lacking one or more DNA repair genes . Our results suggest that the product of the xthA gene, exonuclease III, is required for the repair of lesions induced by SnCl2 . We further investigated the mutagenic properties of SnCl2 to a molecular level by using the supF tRNA gene as target in a forward mutational system . We have found that the survival of E . coli cells was strongly reduced with increasing concentrations of SnCl2 . Moreover, when the shuttle vector pAC189 carrying the supF gene was treated with SnCl2, and then transfected to E . coli, we observed that its transformation efficiency dropped when compared to the non-treated control, with a parallel increase in mutation frequency after the damaged plasmids have replicated in bacterial cells . The mutation spectrum induced by SnCl2 reveals a high frequency of base substitutions, involving guanines . Sequence analysis of 41 independent supF mutant plasmids revealed that 39 mutants contained base substitutions, with 21 G:C to T:A and 17 G:C to C:G transversions . G to T transversions presumably resulted from 8-oxoG . However, the G to C one may be due to a yet unidentified lesion.

Nahrung, 1998 Aug, 42(3-4), 145 - 7
Enzymatic phosphorylation of food proteins by purified and recombinant protein kinase CK2; Chardot T et al.; Protein kinase CK2 formerly called casein kinase II is a protein kinase able to phosphorylate more than 100 proteic substrates . We have purified protein kinase CK2 from the yeast Y . lipolytica to phosphorylate milk and plant reserve proteins to a significant extent . In the case of plant reserve proteins, which are polymeric substrates, not all subunits are substrate for protein kinase CK2, even if non phosphorylated subunits contain significant potent phosphorylations sites . Best substrates were soy beta-conglycinin (0.72 P/mol) and dephosphorylated caseins (0.5 P/mol) . We have studied some functional properties of phosphorylated caseins . Solubility was improved for all pH values but pI . Sensitivity to calcium has also been assessed, and it is slightly improved upon phosphorylation . We have cloned the catalytic subunit of protein kinase CK2 from yeast Y . lipolytica . The recombinant catalytic subunit expressed in E . coli was active and displayed kinetic properties similar to those of the purified enzyme . The recombinant catalytic subunit was able to phosphorylate plant reserve proteins and milk proteins to a significant extent . Best substrates were soy beta-conglycinin (1.0 P/mol), and glycinin (0.59 P/mol).

Nahrung, 1998 Aug, 42(3-4), 135 - 8
Engineering of trypsin and its impact on beta-casein processing; Chobert JM et al.; Tryptic processing of beta-casein yields several important nutraceutic and nutritious peptides . However, a final product peptide (1-105) stops the processing, inhibiting the enzyme . In attempt to modulate catalytic properties of this protease, K188 was replaced with aromatic amino acid residues . This aimed amplification of local hydrophobic and electrostatic interactions at the substrate binding site . The catalytic properties of obtained mutants (K188F, K188Y, and K188W) were measured at pH 7, 8, 9, and 10 with synthetic substrates and beta-casein . Kinetic analysis revealed that all the mutants conserve the capacity to split peptide bonds involving arginyl and lysyl residues . However, depending on mutation, the optimum pH of activity changes . As shown only by proteolysis of a natural substrate, produced mutants cleaved near 30 new peptide bonds compared to wild-type trypsin, 8 of them involving asparagine and glutamine amino acids . Some of the new cleavage sites can be related to the nature of the amino acid residue introduced in position 188 . Consequently, only the joint use of several methods (synthetic substrate, protein substrate, influence of pH) can help to define better the differences of catalytic properties of wild-type and mutant proteases . Modifications introduced by the mutations are at the origin of the alteration of the specificity of the studied enzymes which are cleaving beta-casein in many places, hydrolysing well the fragment 1-105 . Since many tryptic inhibitors contain amidated Glu and Asp, and form amyloid structures, the new mutants could be used in hydrolysing resistant proteic structures.

Biochem Mol Biol Int, 1998 Aug, 45(5), 871 - 8
Enzymatic methylation of recombinant TIS21 protein-arginine residues; Lim IK et al.; Recombinant TIS21 protein was overexpressed in Escherichia coli harboring the expression vector plasmid pQE-30 carrying the TIS21 cDNA coding sequence containing an extra 120 nucleotides upstream . Employing this protein consisting of 158 amino acid residues of the main chain plus 40 residues of the fusion peptide . It was found that one of the protein methylase I group {S-adenosylmethionine:nuclear protein/histone-arginine N-methyltransferase; BC 2.1.1.23; J . Biol . Chem., 269, 1075 (1994)} methylated this protein . The methylation products were identified as guanidino-N-methylated arginines . Some of the kinetics of the reaction are described.

Mech Dev, 1998 Jul, 75(1-2), 117 - 26
Mesodermal cell fate decisions in Drosophila are under the control of the lineage genes numb, Notch, and sanpodo; Park M et al.; In Drosophila, much has been learned about the specification of neuronal cell fates but little is known about the lineage of mesodermal cells with different developmental fates . Initially in development, individual mesodermal precursor cells are singled out to become the founder cells for specific muscles . The selection of muscle founder cells is thought to employ a Notch-mediated process of lateral inhibition, similar to what is observed for the specification of neural precursors . These muscle founder cells then seem to fuse with the surrounding, uncommitted myocytes inducing the formation of muscle fiber syncytia . In contrast, the differentiated progeny of neural precursor cells are usually the result of a fixed pattern of asymmetric cell divisions which are directed, in part, by interactions between numb, a localized intracellular-receptor protein, sanpodo (spdo), a potential tropomodulin homolog, and Notch, a transmembrane receptor protein . Here, we have investigated the role of these neural lineage genes in the cell fate specification of muscle and heart precursors . In particular, we have focused on a progenitor cell that is likely to produce a mixed lineage, generating both a pericardial heart cell and a somatic muscle founder cell . We show that the asymmetric segregation of Numb into one of these daughter cells antagonizes the function of Notch and spdo by preventing the presumptive muscle founder from assuming the same fate as its cardiac sibling . Our results suggest that asymmetric cell divisions, in addition to the previously-documented inductive mechanisms, play a major role in cardiac and somatic muscle patterning and that additionally the cytoskeleton may have a role in the asymmetrical localization of cell fate determinants .

Structure, 1998 Aug 15, 6(8), 1047 - 55
Involvement of the C terminus in intramolecular nitrogen channeling in glucosamine 6-phosphate synthase: evidence from a 1.6 A crystal structure of the isomerase domain; Teplyakov A et al.; BACKGROUND: Glucosamine 6-phosphate synthase (GlmS) catalyses the first step in hexosamine metabolism, converting fructose-6P (6 phosphate) into glucosamine-6P using glutamine as a nitrogen source . GlmS is a bienzyme complex consisting of two domains that catalyse glutamine hydrolysis and sugar-phosphate isomerisation, respectively . Knowledge of the three-dimensional structure of GlmS is essential for understanding the general principles of catalysis by ketol isomerases and the mechanism of nitrogen transfer in glutamine amidotransferases . RESULTS: The crystal structure of the isomerase domain of the Escherichia coli GlmS with the reaction product, glucosamine-6P, has been determined at 1.57 A resolution . It is comprised of two topologically identical subdomains, each of which is dominated by a nucleotide-binding motif of a flavodoxin type . The catalytic site is assembled by dimerisation of the protein . CONCLUSIONS: The isomerase active site of GlmS seems to be the result of evolution through gene duplication and subsequent dimerisation . Isomerisation of fructose-6P is likely to involve the formation of a Schiff base with Lys603 of the enzyme, the ring-opening step catalysed by His504, and the proton transfer from C1 to C2 of the substrate effected by Glu488 . The highly conserved C-terminal fragment of the chain may play a key role in substrate binding, catalysis and communication with the glutaminase domain . The corresponding sequence pattern DXPXXLAK{SC}VT (in single-letter amino-acid code, where X is any amino acid and letters in brackets indicate that either serine or cysteine may take this position) may be considered as a fingerprint of GlmS.

Structure, 1998 Aug 15, 6(8), 993 - 1005
The solution structure of an RNA loop-loop complex: the ColE1 inverted loop sequence; Lee AJ et al.; BACKGROUND: Replication of the ColE1 plasmid of Escherichia coli is regulated by the interaction of sense and antisense plasmid-encoded transcripts . The antisense RNA I negatively regulates the replication of the plasmid by duplex formation with complementary RNA II . The interaction is initiated by the formation of a double helix between seven-nucleotide loops from each RNA and is stabilized by binding of the RNA one modulator (ROM) protein . The ROM protein is thought to recognize a specific RNA structure, regardless of sequence . RESULTS: The solution structure of a loop-loop complex between model RNA hairpins that resemble RNA I and RNA II has been determined by nuclear magnetic resonance spectroscopy . The model hairpins have loop sequences inverted 5' to 3' relative to the wild-type sequence and were chosen because of their complex's slow dissociation in comparison to the wild type . The complex has continuous stacking from the 3'-side of one stem helix through the loop-loop helix to the other stem helix . One residue from each hairpin has a unique phosphodiester bond which bridges and narrows the major groove . These bridging phosphates are in close proximity to the phosphate groups of the adjacent bases, forming unique structural motifs called phosphate clusters . The purine residue at the 3'-end of the loop-loop helix of one RNA stacks on a purine residue on the 5'-side of the other RNA stem, and there are strong cross-strand stacking interactions between guanine bases in the stem helices adjacent to the loops . CONCLUSIONS: Unique distortions, such as the strong bend and the phosphate clusters flanking the major groove of the loop-loop helix, provide an attractive nonsequence-specific structural feature for recognition by the ROM protein . The structure provides a basis for rationalizing the sequence dependence of the stability of loop-loop interaction.

Nature, 1998 Sep 3, 395(6697), 93 - 6
RNA polymerase II is an essential mRNA polyadenylation factor; Hirose Y et al.; Production of messenger RNA in eukaryotic cells is a complex, multistep process . mRNA polyadenylation, or 3' processing, requires several protein factors, including cleavage/polyadenylation-specificity factor (CPSF), cleavage-stimulation factor, two cleavage factors and poly(A) polymerase . These proteins seem to be unnecessary for other steps in mRNA synthesis such as transcription and splicing, and factors required for these processes were not considered to be essential for polyadenylation . Nonetheless, these reactions may be linked so that they are effectively coordinated in vivo . For example, the CTD carboxy-terminal domain of the largest subunit of RNA polymerase II (RNAP II) is required for efficient splicing and polyadenylation in vivo, and CPSF is brought to a promoter by the transcription factor TFIID and transferred to RNAP II at the time of transcription initiation . These findings suggest that polyadenylation factors can be recruited to an RNA 3'-processing signal by RNAP II, where they dissociate from the polymerase and initiate polyadenylation . Here we present results that extend this model by showing that RNAP II is actually required, in the absence of transcription, for 3' processing in vitro.

J Mol Biol, 1998 Sep 25, 282(3), 495 - 504
CAP, the -45 region, and RNA polymerase: three partners in transcription initiation at lacP1 in Escherichia coli; Noel RJ Jr et al.; The lac operon of Escherichia coli is positively regulated by the catabolite activator protein (CAP) bound upstream of the -45 region (CAP binding is centered at -61.5; the -45 region extends from -50 to -38) . Certain mutations within the -45 region generate sequences that resemble UP elements in base composition and mimic the stimulation by the rrnBP1 UP element, yielding up to 15-fold stimulation in vivo . These -45 region "UP mutants" are compromised in their CAP stimulation . CAP and UP elements do not act in a fully additive manner in vivo at the lac operon . Transcription assays with the wild-type lac promoter and an UP mutant of lac indicate that CAP and UP DNA also fail to act in a completely additive manner in vitro . RNA polymerase can stabilize CAP binding to promoter DNA with a -45 region UP element against a heparin challenge . This shows that CAP and the UP DNA do not compete for the alpha-CTD as a mechanism for their lack of additivity . CAP and UP elements both demonstrate decreased stimulation of transcription as RNA polymerase concentration is increased from 0.05 to 10 nM in in vitro transcription experiments . In addition CAP also stimulates transcription in a manner that does not decrease as RNA polymerase is varied over this concentration range . This invariable stimulation is by two- to threefold and occurs both in vivo and in vitro . It is not dependent upon the alpha-CTD of RNA polymerase and is maintained in the presence of the AR1 CAP mutant HL159 . This two- to threefold invariable CAP stimulation appears to depend on the -45 region sequence as our -45 region mutants demonstrate different responses to HL159 CAP stimulation in vivo .

FEBS Lett, 1998 Aug 14, 433(1-2), 153 - 6
Conformational analysis of the interdomain linker of the central homology region of chloroplast initiation factor IF3 supports a structural model of two compact domains connected by a flexible tether; Hua Y et al.; A peptide corresponding to the interdomain linker of chloroplast IF3 has been synthesized and its structure studied by NMR and CD as a function of temperature and pH . At low temperature and neutral pH the apparent helical content is 25% . pH and ionic strength dependent CD studies demonstrate that sidechain-sidechain interactions stabilize the structure observed at low temperature . The helicity decreases with temperature and above 25 degrees C the peptide is less than 15% helical . These results indicate that the peptide has little intrinsic tendency to form helical structure at physiologically relevant temperatures and strongly suggests that the linker region is flexible in intact chloroplast IF3.

FEBS Lett, 1998 Aug 14, 433(1-2), 143 - 8
cDNA cloning and characterization of mouse nifS-like protein, m-Nfs1: mitochondrial localization of eukaryotic NifS-like proteins; Nakai Y et al.; We have isolated a mouse cDNA which shows significant sequence similarity to the yeast nifS-like gene (y-NFS1), and termed it m-Nfs1 . The deduced protein sequence (459 amino acids long) has several characteristic features common to those of bacterial NifS proteins, but distinct from them by its amino-terminal extension which contains a typical mitochondrial targeting presequence . m-Nfs1 was found to be a soluble 47-kDa protein in the matrix fraction of mouse liver mitochondria . The m-Nfs1 gene was ubiquitously expressed in most tissues, suggesting its housekeeping function in vivo . We also found that the gamma-NFS1 protein was localized in the mitochondrial matrix in yeast cells . These results suggest that both eukaryotic NifS-like proteins may play some roles in mitochondrial functions.

FEBS Lett, 1998 Aug 14, 433(1-2), 132 - 8
Acylation of Streptomyces type II polyketide synthase acyl carrier proteins; Crosby J et al.; Acyl derivatives of type II PKS ACPs are required for in vitro studies of polyketide biosynthesis . The presence of an exposed cysteine residue prevented specific chemical acylation of the phosphopantetheine thiol of the actinorhodin PKS holo ACP . Acylation studies were further complicated by intramolecular disulphide formation between cysteine 17 and the phosphopantetheine . The presence of this intramolecular disulphide was confirmed by tryptic digestion of the ACP followed by ESMS analysis of the fragments . An act Cys17Ser ACP was engineered by site-directed mutagenesis . S-Acyl adducts of act C17S, oxytetracycline and griseusin holo ACPs were rapidly formed by reaction with hexanoyl, 5-ketohexanoyl and protected acetoacetyl imidazolides . Comparisons with type 11 FAS ACPs were made.

FEBS Lett, 1998 Aug 14, 433(1-2), 83 - 8
Anchoring antibodies to membranes using a diphtheria toxin T domain-ZZ fusion protein as a pH sensitive membrane anchor; Nizard P et al.; We have constructed a fusion protein, T-ZZ, in which the IgG-Fc binding protein ZZ was fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain) . While soluble at neutral pH, T-ZZ retained the capacity of the T domain to bind to phospholipid membranes at acidic pH . Once anchored to the membrane, the ZZ part of the protein was capable of binding mouse monoclonal or rabbit polyclonal IgG . Our results show that the T-ZZ protein can function as a pH sensitive membrane anchor for the linkage of IgG to the membrane of lipid vesicles, adherent and non-adherent cells.

Eur J Biochem, 1998 Aug 1, 255(3), 746 - 54
Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit--identification of a soluble precursor of the small subunit in a hypB mutant; Sargent F et al.; An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the periplasmic face of the cytoplasmic membrane, and the large and small subunits N-terminally sequenced . The large subunit is encoded by the hybC gene and shows no N-terminal processing, other than removal of the initiator methionine during its biosynthesis . Both N-terminal and the subsequent internal tryptic-fragment amino acid sequence indicate that the small subunit is neither encoded by hybA, a gene previously identified as encoding the small subunit {Menon et al . (1994) J . Bacteriol . 176, 4416-4423}, nor any of the remaining genes in the hyb operon . Genome sequence analysis revealed the presence of an open reading frame which could potentially encode the peptide sequences of the proteolysed small subunit . The gene, designated hyb0, lies directly upstream of, and is separated by two nucleotides from, the start of the hybA gene . Hyb0, which shares an approximate 40% identity with other hydrogenase small subunit amino acid sequences, is synthesised with an N-terminal signal sequence containing a twin-arginine motif which is probably required for export of the enzyme . In the mature enzyme the small subunit is proteolytically cleaved after Ala37 . Immunological analysis of strains overproducing either recombinant Hyb0 or HybA using antibodies specific for hydrogenase 2, readily identified Hyb0 as the small subunit . In a pleiotropic hypB mutant, which is unable to insert nickel into the active site, both the large and small subunits accumulate as unprocessed, soluble forms, consistent with the two subunits being assembled and processed in a coordinated manner during biosynthesis.

Eur J Biochem, 1998 Aug 1, 255(3), 739 - 45
Identification of major wheat allergens by means of the Escherichia coli expression system; Maruyama N et al.; Wheat proteins were fractionated into salt-soluble, glutenin-rich, and gliadin-rich fractions . Reactivities of these protein fractions with sera of patients with wheat-associated allergies were examined under various conditions . The relative reactivity of the fractions was generally in the order glutenin-rich > gliadin-rich >> salt-soluble fractions, although their reactivities were variable among patients and among the reaction conditions, indicating that the kind, the number and the epitope of allergens were variable among patients . To identify major allergens, alpha-, gamma- and omega-gliadin, and low-molecular-mass (LMM)- and high-molecular-mass (HMM)-glutenin genes were expressed in Escherichia coli by means of a pET vector . Recombinant gliadins and glutenins were partially purified on the basis of the solubilities of prolamin and glutelin . The partially purified recombinant proteins were reacted with the patients' sera . LMM glutenin containing many Gln-Gln-Gln-Pro-Pro motifs, which was identified to be IgE-binding epitope {Tanabe, S., Arai, S., Yanagihara, Y., Mita, H., Takahashi, K . & Watanabe, M . (1996) Biochem . Biophys . Res . Commun . 219, 290-293}, exhibited the highest reactivity . The next highest reactivities were observed on alpha-gliadin and gamma-gliadin, which had not been identified as allergens.

Eur J Biochem, 1998 Aug 1, 255(3), 654 - 62
The N-terminus of A1-type myosin essential light chains binds actin and modulates myosin motor function; Timson DJ et al.; There are two isoforms (A1 and A2) of the myosin essential light chain (ELC) and consequently two isoenzymes of myosin subfragment 1 (S1), S1(A1) and S1(A2) . The two isoenzymes differ in their kinetic properties with S1(A1) having a lower apparent Km for actin and a slower turnover of MgATP (k(cat)) than S1(A2) . The two forms of the ELC differ only at their N-termini where A1 has an additional 40-odd amino acids that are not present in A2 . The human atrial ELC (an A1-type ELC) was overexpressed in Escherichia coli and purified by ammonium sulphate fractionation and ion-exchange chromatography . The recombinant ELC had actin-activated MgATPase kinetics similar to those for rabbit skeletal S1(A1) under the same conditions . Deletion of the first 45 amino acid residues resulted in an ELC similar to the rabbit skeletal A2 isoform and, when hybridised into S1, in S1(A2)-like kinetic properties . Results obtained with an ELC mutant that lacks the first 11 residues were intermediate between these two extremes but tending towards the S1(A2)-like phenotype . The wild-type ELC (both hybridised into S1 or free in solution) could be cross-linked to F-actin, whereas the deletion mutant lacking the first 45 amino acids could not . The deletion mutant lacking the first 11 amino acids cross-linked only poorly under the same conditions, consistent with the MgATPase data . We therefore conclude that these N-terminal eleven amino acids predominantly encode an actin-binding site which modulates the kinetics of the myosin motor . Furthermore, while free A1-type ELC cross-linked to both polymeric F-actin and the monomeric G-actin:DNase-I complex, the same ELC in S1(A1) could only cross-link to F-actin . This suggests that the light chain binds to a different actin monomer than the heavy chain.

Eur J Biochem, 1998 Aug 1, 255(3), 628 - 37
Purification and characterization of recombinant Thermotoga maritima dihydrofolate reductase; Wilquet V et al.; We have overexpressed the gene for dihydrofolate reductase (DHFR) from Thermotoga maritima in Escherichia coli and characterized the biochemical properties of the recombinant protein . This enzyme is involved in the de novo synthesis of deoxythymidine 5'-phosphate and is critical for cell growth . High levels of T . maritima DHFR in the new expression system conferred resistance to high levels of DHFR inhibitors which inhibit the growth of non-recombinant cells . The enzyme was purified to homogeneity in the following two steps: heat treatment followed by affinity chromatography or cation-exchange chromatography . Most of the biochemical properties of T . maritima DHFR resemble those of other bacterial or eukaryotic DHFRs, however, some are unique to T . maritima DHFR . The pH optima for activity, Km for substrates, and polypeptide chain length of T . maritima DHFR are similar to those of other DHFRs . In addition, the secondary structure of T . maritima DHFR, as measured by circular dichroism, is similar to that of other DHFRs . Interestingly, T . maritima DHFR exhibits some characteristics of eukaryotic DHFRs, such as a basic pI, an excess of positively charged residues in the polypeptide chain and activation of the enzyme by inorganic salts and urea . Unlike most other DHFRs which are monomeric or part of a bifunctional DHFR-thymidylate synthase (TS) enzyme, T . maritima DHFR seems to generally form a dimer in solution and is also much more thermostable than other DHFRs . It may be that dimer formation is a key factor in determining the stability of T . maritima DHFR.

FEBS Lett, 1998 Aug 28, 434(1-2), 160 - 4
Inhibitory effect of acidic pH on OmpC porin: wild-type and mutant studies; Liu N et al.; By use of the patch clamp technique, we have compared the electrophysiological signature of OmpC porin channels at neutral and acidic pH . The perfusion of pH 5.4 buffer to the periplasmic side of excised patches promoted the closure or block of approximately 20% of the open porins present in the patch without changes in their single channel conductance . Besides this effect on the main, long-lived open state, lowering the pH also suppressed the spontaneous transitions of channels to another distinct short-lived open state . The inhibitory effect on the opening kinetics was particularly visible in two mutants (K16Q and E109Q) in which transitions to the short-lived open state are enhanced by the mutations themselves at pH 7.2 . On the other hand, the R124Q mutant responded to acidic pH by an increased gating to the short-lived open state . The results suggest that acidic pH stabilizes a closed state of OmpC porin, and that the pH sensitivity might be conferred in part by R124, but not by K16 or E109.

FEBS Lett, 1998 Aug 28, 434(1-2), 57 - 60
ATP synthesis by the F1Fo ATP synthase of Escherichia coli is obligatorily dependent on the electric potential; Kaim G et al.; The H+-translocating F1Fo ATP synthase of Escherichia coli was purified and reconstituted into proteoliposomes . This system catalyzed ATP synthesis when energized by an acid/base transition (pHin = 5.0; pHout = 8.3) with succinate, malonate or maleinate but not with MES as the acidic buffer . Under these experimental conditions an electric potential of 125-130 mV is generated by the diffusion of succinate, probably the monoanionic species, whereas with MES buffer the measured potential was at background level (approximately 5 mV) . ATP was also synthesized at pH 7.2 in the absence of a delta pH by applying a K+/valinomycin diffusion potential . The rate of ATP synthesis increased with the potential in an exponential manner with an inflection point at about 70 mV . We conclude from these results that delta pH and delta psi are kinetically unequivalent driving forces for ATP synthesis by the E . coli ATP synthase and that delta psi is a mandatory force for this synthesis . The significance of these findings for the mechanism of ATP synthesis in general is discussed.

FEBS Lett, 1998 Aug 28, 434(1-2), 28 - 32
Expression of pI(Cln) in Escherichia coli gives a strong tolerance to hypotonic stress; Tao GZ et al.; We amplified the coding region DNA sequence from a rat renal pI(Cln) cDNA by PCR and expressed the protein in Escherichia coli cells . The cells were exposed to hypotonic conditions followed by spreading them onto LB plates for subsequent colony survival assay . The present study demonstrated that the cells expressing pI(Cln) exhibit a strong resistance to hypotonic stress . Moreover, the resistance was specifically inhibited by extracellular ATP and some anion channel inhibitors . These findings indicate that the expression of pI(Cln) directly confers tolerance to hypotonic stress, and pI(Cln) is concluded to be an important molecule for cell-volume regulation.

FEBS Lett, 1998 Aug 28, 434(1-2), 23 - 7
Engineering of solvent-exposed loops in Escherichia coli beta-galactosidase; Feliu JX et al.; The Escherichia coli beta-galactosidase is a high molecular mass tetrameric enzyme extensively used as a molecular marker . Despite its proven utility as a partner in fusion proteins, previous attempts to generate insertional mutants rendered inactive or poorly active enzymes, hampering its further engineering for the construction of multifunctional enzymes . We have explored several solvent-exposed loops on the tetramer, namely those spanning residues 246-254, 271-287, 578-584, 770-773, and 793-806, as acceptor sites to accommodate functional protein segments on the surface of active beta-galactosidase enzymes . An RGD-containing antigenic peptide positioned in these sites interacts efficiently with specific monoclonal antibodies as well as target integrins on the surface of mammalian cells . The resulting chimeric enzymes are soluble, stable, produced in high yields and enzymatically active . Moreover, the identified insertion sites could be appropriated for the design of promising beta-galactosidase-based molecular sensors.

Gesundheitswesen, 1998 Jul, 60(7), 420 - 30
{Unconventional diagnostic and therapeutic methods in environmental medicine}; Oepen I; In the sphere of environmental medicine--analogous to other fields like oncology and chronic diseases--not only proven and approved methods, but also unconvential methods are offered, without evidence of efficacy . The application of these methods has the possible consequence of wrong diagnosis and malpractice . Examples are discussed such as Kirlian photography, electroacupuncture according to Voll, bioresonance diagnosis/therapy, kinesiology, regulation therapy according to Rost, "clinical ecology" according to Runow with, among others, the provocation/neutralisation test, a vaccination therapy with E . coli and finally electrosmog as an environmental noxa . Concerning the admissibility of contested methods, statements of medical specialist societies, judgements, and the law of medical products are quoted . In conclusion, the question of the origin of the ideas and alleged results of unconvential medicine is followed up and conclusions are drawn.

J Biol Chem, 1998 Sep 25, 273(39), 25516 - 26
Characterization of the Escherichia coli RNA 3'-terminal phosphate cyclase and its sigma54-regulated operon; Genschik P et al.; The RNA 3'-terminal phosphate cyclase catalyzes the ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of various RNA substrates . Recent cloning of a cDNA encoding the human cyclase indicated that genes encoding cyclase-like proteins are conserved among Eucarya, Bacteria, and Archaea . The protein encoded by the Escherichia coli gene was overexpressed and shown to have the RNA 3'-phosphate cyclase activity (Genschik, P., Billy, E., Swianiewicz, M., and Filipowicz, W . (1997) EMBO J . 16, 2955-2967) . Analysis of the requirements and substrate specificity of the E . coli protein, presented in this work, demonstrates that properties of the bacterial and human enzymes are similar . ATP is the best cofactor (Km = 20 microM), whereas GTP (Km = 100 microM) and other nucleoside triphosphates (NTPs) act less efficiently . The enzyme undergoes nucleotidylation in the presence of {alpha-32P}ATP and, to a lesser extent, also in the presence of other NTPs . Comparison of 3'-phosphorylated oligoribonucleotides and oligodeoxyribonucleotides of identical sequence demonstrated that the latter are at least 300-fold poorer substrates for the enzyme . The E . coli cyclase gene, named rtcA, forms part of an uncharacterized operon containing two additional open reading frames (ORFs) . The ORF positioned immediately upstream, named rtcB, encodes a protein that is also highly conserved between Eucarya, Bacteria, and Archaea . Another ORF, called rtcR, is positioned upstream of the rtcA/rtcB unit and is transcribed in the opposite direction . It encodes a protein having features of sigma54-dependent regulators . By overexpressing the N-terminally truncated form of RtcR, we demonstrate that this regulator indeed controls expression of rtcA and rtcB in a sigma54-dependent manner . Also consistent with the involvement of sigma54, the region upstream of the transcription start site of the rtcA/rtcB mRNA contains the -12 and -24 elements, TTGCA and TGGCA, respectively, characteristic of sigma54-dependent promoters . The cyclase gene is nonessential as demonstrated by knockout experiments . Possible functions of the cyclase in RNA metabolism are discussed.

J Biol Chem, 1998 Sep 25, 273(39), 25310 - 9
Forced expression of Id-1 in the adult mouse small intestinal epithelium is associated with development of adenomas; Wice BM et al.; Ids are dominant-negative helix-loop-helix (HLH) proteins that play overlapping yet distinct roles in antagonizing basic HLH transcription factors . Although Ids affect myogenesis, neurogenesis, and B-cell development, little is known about their in vivo functions in epithelia . We have examined the effects of forced expression of Id-1 in the small intestinal epithelium of adult chimeric mice . 129/Sv embryonic stem cells, transfected with DNA containing Id-1 under the control of transcriptional regulatory elements that function in all intestinal epithelial cell lineages, were introduced into C57Bl/6 (B6) blastocysts heterozygous for the ROSA26 marker . The B6 ROSA26/+ intestinal epithelium of the resulting adult chimeras produces Escherichia coli beta-galactosidase, allowing identification of this internal control cell population . Chimeras produced from nontransfected embryonic stem cells served as additional controls . Immunohistochemical studies of the control chimeras indicated that the small intestinal epithelium supports a complex pattern of endogenous Id expression . Id-1 is restricted to the cytoplasm; levels do not decrease as descendants of multipotent intestinal stem cells differentiate . Id-2 and Id-3 are only detectable in nuclei; levels increase markedly as epithelial cells differentiate . Forced expression of Id-1 in the 129/Sv epithelium results in a decline in Id-2 and Id-3 to below the limits of immunodetection . A subset of chimeric-transgenic mice lacked growth factor- and defensin-producing Paneth cells in their 129/Sv epithelium and also developed intestinal adenomas . These changes were not present in normal control chimeras . Adenomas were composed of proliferating beta-Gal-positive and -negative epithelial cells, suggesting that they arose through cooperative interactions between 129/Sv(Id-1) and B6 ROSA26/+ cells . These chimeras provide a model for studying how perturbations in Id expression affect tumorigenesis.

J Biol Chem, 1998 Sep 25, 273(39), 25148 - 57
Murine HIP/L29 is a heparin-binding protein with a restricted pattern of expression in adult tissues; Hoke DE et al.; Heparin/heparan sulfate (Hp/HS)-binding proteins are implicated in a variety of cell biological processes including cell adhesion, modulation of blood coagulation, and cytokine/growth factor action . Hp/HS-interacting protein (HIP) has been identified in various adult tissues in humans . HIP supports high affinity, selective binding to Hp/HS, promotes cell adhesion, and modulates blood coagulation activities via Hp/HS-dependent mechanisms . Herein, a murine ortholog of human HIP is described that is 78.8% identical to human HIP and 99.8% identical at the cDNA level and identical at the amino acid level to a previously described murine ribosomal protein, L29 . Western blot analyses and immunohistological staining with affinity-purified antibodies generated against two distinct peptide sequences of murine HIP/L29 indicate that HIP/L29 is differentially expressed in adult murine tissues and cell types . In the normal murine mammary epithelial cell line, NMuMG, HIP/L29 is enriched in the 100,000 x g particulate fraction . HIP/L29 can be solubilized from the 100,000 x g particulate fraction with 0.8 M NaCl, suggesting that it is a peripheral membrane protein . HIP/L29 directly binds 125I-Hp in gel overlay assays and requires 0.75 M NaCl for elution from Hp-agarose . In addition, recombinant murine HIP expressed in Escherichia coli binds Hp in a saturable and highly selective manner, compared with other glycosaminoglycans including dermatan sulfate, chondroitin sulfate, keratan sulfate, and hyaluronic acid . Collectively, these data indicate that murine HIP/L29, like its human ortholog, is a Hp-binding protein expressed in a restricted manner in adult tissues.

J Mol Biol, 1998 Sep 25, 282(3), 571 - 83
Cointegrase, a naturally occurring, truncated form of IS21 transposase, catalyzes replicon fusion rather than simple insertion of IS21; Schmid S et al.; The bacterial insertion sequence IS21 contains two genes, istA and istB, which are organized as an operon . IS21 spontaneously forms tandem repeats designated (IS21)2 . Plasmids carrying (IS21)2 react efficiently with other replicons, producing cointegrates via a cut-and-paste mechanism . Here we show that transposition of a single IS21 element (simple insertion) and cointegrate formation involving (IS21)2 result from two distinct non-replicative pathways, which are essentially due to two differentiated IstA proteins, transposase and cointegrase . In Escherichia coli, transposase was characterized as the full-length, 46 kDa product of the istA gene, whereas the 45 kDa cointegrase was expressed, in-frame, from a natural internal translation start of istA . The istB gene, which could be experimentally disconnected from istA, provided a helper protein that strongly stimulated the transposase and cointegrase-driven reactions . Site-directed mutagenesis was used to express either cointegrase or transposase from the istA gene . Cointegrase promoted replicon fusion at high frequencies by acting on IS21 ends which were linked by 2, 3, or 4 bp junction sequences in (IS21)2 . By contrast, cointegrase poorly catalyzed simple insertion of IS21 elements . Transposase had intermediate, uniform activity in both pathways . The ability of transposase to synapse two widely spaced IS21 ends may reside in the eight N-terminal amino acid residues which are absent from cointegrase . Given the 2 or 3 bp spacing in naturally occurring IS21 tandems and the specialization of cointegrase, the fulminant spread of IS21 via cointegration can now be understood .

Biochemistry, 1998 Sep 15, 37(37), 13011 - 20
Thermodynamics and kinetics of the reaction of a single-chain antibody fragment (scFv) with the leucine zipper domain of transcription factor GCN4; Weber-Bornhauser S et al.; Single-chain Fv (scFv) fragments of antibodies have become important analytical and therapeutic tools in biology and medicine . The reaction of scFv fragments has not been well-characterized with respect to the energetics and kinetics of antigen binding . This paper describes the thermodynamic and kinetic behavior of the high-affinity scFv fragment SW1 directed against the dimeric leucine zipper domain of the yeast transcription factor GCN4 . The scFv fragment was selected by the phage display technique from the immune repertoire of a mouse that had been immunized with the leucine zipper domain of GCN4 . The scFv fragment was produced in high yield in Escherichia coli inclusion bodies and refolded from the denatured state . Differential scanning calorimetry showed that SW1 was stable up to about 50 degreesC, but the subsequent thermal denaturation was irreversible (Tm approximately 68 degreesC) . The scFv fragment specifically recognized the dimeric leucine zipper conformation . Two scFv fragments bound to the GCN4 dimer to form the complex (scFv)2-GCN4 . Because of its repetitive structure, the rod-shaped GCN4 leucine zipper may present two similar epitopes for the scFv fragment . Surprisingly, the binding reaction was highly cooperative, that is, the species (scFv)2-GCN4 dominated over scFv-GCN4 even in the presence of a large excess of the antigen GCN4 . It is speculated that cooperativity resulted from direct interaction between the two GCN4-bound scFv fragments . At 25 degreesC, the average binding enthalpy for a scFv fragment was favorable (-61 kJ mol-1), the entropy change was unfavorable, and the change in heat capacity was -1.27 +/- 0.14 kJ mol-1 K-1 . As a result of enthalpy-entropy compensation, the free binding energy was virtually independent of temperature in the physiological temperature range . Antigen binding in solution could be described by a single-exponential reaction with an apparent rate constant of 1 x 10(6) M-1 s-1 . Binding followed in a biosensor with the dimeric GCN4 coupled to the surface of the metal oxide sensor chip was 20 times slower.

Biochemistry, 1998 Sep 15, 37(37), 12818 - 28
Stability of recombinant yeast protoporphyrinogen oxidase: effects of diphenyl ether-type herbicides and diphenyleneiodonium; Arnould S et al.; Protoporphyrinogen oxidase catalyzes the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX and is the molecular target of diphenyl ether-type herbicides . Structural features of yeast protoporphyrinogen oxidase were assessed by circular dichroism studies on the enzyme purified from E . coli cells engineered to overproduce the protein . Coexpression of the bacterial gene ArgU that encodes tRNAAGA,AGG and a low induction temperature for protein synthesis were critical for producing protoporphyrinogen oxidase as a native, active, membrane-bound flavoprotein . The secondary structure of the protoporphyrinogen oxidase was 40.0 +/- 1 . 5% alpha helix, 23.5 +/- 2.5% beta sheet, 18.0 +/- 2.0% beta turn, and 18.5 +/- 2.5% random-coil . Purified protoporphyrinogen oxidase appeared to be a monomeric protein that was relatively heat-labile (Tm of 44 +/- 0.5 degreesC) . Acifluorfen, a potent inhibitor that competes with the tetrapyrrole substrate, and to a lower extent FAD, the cofactor of the enzyme, protected the protein from thermal denaturation, raising the Tm to 50.5 +/- 0.5 degreesC (acifluorfen) and 46.5 +/- 0.5 degreesC (FAD) . However, diphenyleneiodonium, a slow tight-binding inhibitor that competes with dioxygen, did not protect the enzyme from heat denaturation . Acifluorfen binding to the protein increased the activation energy for the denaturation from 15 to 80 kJ.mol-1 . The unfolding of the protein was a two-step process, with an initial fast reversible unfolding of the native protein followed by slow aggregation of the unfolded monomers . Functional analysis indicated that heat denaturation caused a loss of enzyme activity and of the specific binding of radiolabeled inhibitor . Both processes occurred in a biphasic manner, with a transition temperature of 45 degreesC.

Biochemistry, 1998 Sep 15, 37(37), 12802 - 10
The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state; Jiang P et al.; The regulation of Escherichia coli glutamine synthetase (GS) by reversible adenylylation has provided one of the classical paradigms for signal transduction by cyclic cascades . Yet, many mechanistic features of this regulation remain to be elucidated . We examined the regulation of GS adenylylation state in a reconstituted system containing GS, adenylyltransferase (ATase), the PII signal transduction protein that controls ATase, and the uridylyltransferase/uridylyl-removing enzyme (UTase/UR), which has a role in regulating PII . In this reconstituted bicyclic cascade system, the adenylylation state of GS was regulated reciprocally by the small molecule effectors 2-ketoglutarate and glutamine at physiological effector concentrations . By examination of the individual regulatory monocycles and comparison to the bicyclic system and existing data, we could deduce that the only sensors of 2-ketoglutarate were PII and PII-UMP . At physiological conditions, we observed that the main role of 2-ketoglutarate in bringing about the deadenylylation of GS was to inhibit GS adenylylation, and this was due to the allosteric regulation of PII activity . Glutamine acted as an allosteric regulator of both ATase and UTase/UR . We also compared the regulation of GS adenylylation state to the regulation of phosphorylation state of the transcription factor NRI (NtrC) in a reconstituted bicyclic system containing NRI, the bifunctional kinase/phosphatase NRII (NtrB), PII, and the UTase/UR . This comparison indicated that, at a fixed 2-ketoglutarate concentration, the regulation of GS adenylylation state by glutamine was sharper and occurred at a higher concentration than did the regulation of NRI phosphorylation . The possible biological implications of this regulatory arrangement are discussed.

Biochemistry, 1998 Sep 15, 37(37), 12795 - 801
Reconstitution of the signal-transduction bicyclic cascade responsible for the regulation of Ntr gene transcription in Escherichia coli; Jiang P et al.; Nitrogen-regulation of gene transcription in Escherichia coli results from the regulation of the phosphorylation state of the enhancer-binding transcription factor NRI (NtrC) . We examined the regulation of NRI phosphorylation in a reconstituted bicyclic cascade system containing four regulatory proteins: NRI, the signal-transducing uridylyltransferase/uridylyl-removing enzyme (UTase/UR), its substrate the signal transduction protein PII, and the kinase/phosphatase NRII (NtrB), which is a PII receptor that phosphorylates and dephosphorylates NRI . In this reconstituted system, the phosphorylation state of NRI was regulated reciprocally by the small molecule effectors glutamine, which prevented the accumulation of NRI-P, and 2-ketoglutarate, which caused accumulation of NRI-P . Regulation of the bicyclic system by glutamine was exclusively due to sensation and signal-transduction by the UTase/UR-PII monocycle, which was observed to function essentially as a glutamine-sensing apparatus . In contrast, regulation of NRI phosphorylation by 2-ketoglutarate, which binds to PII, was due to direct regulation of the NRII-PII interaction and the rate of NRI-P dephosphorylation . Thus, the PII protein transduces the glutamine signal to the NRII-NRI monocycle in the form of its uridylylation state and is also the receptor of the antagonistic 2-ketoglutarate signal, which blocks the activity of unmodified PII.

Biochemistry, 1998 Sep 15, 37(37), 12782 - 94
Enzymological characterization of the signal-transducing uridylyltransferase/uridylyl-removing enzyme (EC 2.7.7.59) of Escherichia coli and its interaction with the PII protein; Jiang P et al.; The uridylyltransferase/uridylyl-removing enzyme (UTase/UR) of Escherichia coli plays an important role in the regulation of nitrogen assimilation by controlling the uridylylation state of the PII signal transduction protein (PII) in response to intracellular signals . The reversible uridylylation of PII indirectly controls the activity of PII receptors that regulate transcription from nitrogen-regulated promoters and the activity of glutamine synthetase . Here, we present a detailed analysis of the uridylyltransferase and uridylyl-removing activities and their regulation by the small molecule effectors ATP, 2-ketoglutarate, and glutamine . Several important features of enzyme mechanism and regulation were elucidated . Mg2+ appeared to be the physiologically relevant metal ion cofactor for both transferase and uridylyl-removing activities . The transferase reaction proceeded by an ordered bi-bi kinetic mechanism, with PII binding before UTP and pyrophosphate (PPi) released before PII-UMP . The uridylyl-removing reaction proceeded with rapid equilibrium binding of substrate and random release of products . Both reactions were activated by ATP and 2-ketoglutarate, which did so by binding only to PII and PII-UMP . The binding of these effectors to PII and PII-UMP was characterized . Glutamine inhibited the transferase reaction by inhibiting the chemistry step, while glutamine provided nonessential mixed-type activation of the uridylyl-removing activity, lowering the apparent Km and increasing kcat . Our data were consistent with the hypothesis that all effects of glutamine are due to the binding of central complexes at a single glutamine site . By comparing the effects of the activators with their reported in vivo concentrations, we conclude that in intact cells the uridylylation state of PII is regulated mainly by the glutamine concentration and is largely independent of the 2-ketoglutarate concentration . Our kinetic data were consistent with the hypothesis that both transferase and uridylyl-removal reactions occurred at a single active center on the enzyme.

Biochemistry, 1998 Sep 15, 37(37), 12753 - 60
A loop deletion in the plant acetohydroxy acid isomeroreductase homodimer generates an active monomer with reduced stability and altered magnesium affinity; Wessel PM et al.; Plant acetohydroxy acid isomeroreductase is a stable homodimer which catalyzes in the presence of magnesium an alkyl migration followed by a NADPH-dependent reduction . Since the enzyme exhibits no kinetic cooperativity either for its cofactor (NADPH and magnesium) or for its substrates, the reason for dimerization of this enzyme was not obvious . Recently, crystallographic studies {Biou, V., et al . (1997) EMBO J . 16, 3405-3415} revealed that the loop of residues 422-431 plays a major part in the dimer interface . To understand the role of the quaternary structure of the enzyme, we have deleted residues 423-430 and substituted Phe 431 for serine . This mutant was further overproduced in Escherichia coli, purified to homogeneity, and characterized . Gel filtration and thermodynamic experiments disclosed that this mutant behaves as an active monomer with reduced thermal stability . Furthermore, kinetic and fluorescence experiments showed that the behavior of the monomer with respect to magnesium was greatly altered . These results demonstrate the function of the quaternary structure of plant acetohydroxy acid isomeroreductase in the stabilization of the tertiary structure but also in the stabilization of a high-affinity magnesium binding site.

Biochemistry, 1998 Sep 15, 37(37), 12744 - 52
Isolation and characterizations of quinone analogue-resistant mutants of bo-type ubiquinol oxidase from Escherichia coli; Sato-Watanabe M et al.; Cytochrome bo is a member of the heme-copper terminal oxidase superfamily and serves as a four-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli . To probe the location and structural properties of the ubiquinol oxidation site, we isolated and characterized five or 10 spontaneous mutants resistant to either 2,6-dimethyl-1,4-benzoquinone, 2,6-dichloro-4-nitrophenol, or 2,6-dichloro-4-dicyanovinylphenol, the potent competitive inhibitors for the oxidation of ubiquinol-1 {Sato-Watanabe, M., Mogi, T., Miyoshi, H., Iwamura, H., Matsushita, K., Adachi, O., and Anraku, Y . (1994) J . Biol . Chem . 269, 28899-28907} . Analyses of the growth yields and the ubiquinol-1 oxidase activities of the mutant membranes showed that the mutations increased the degree of the resistance to the selecting compounds . Notably, several mutants showed the cross-resistance . These data indicate that the binding sites for substrate and the competitive inhibitors are partially overlapped in the ubiquinol oxidation site . All the mutations were linked to the expression vector, and 23 mutations examined were all present in the C-terminal hydrophilic domain (Pro96-His315) of subunit II . Sequencing analysis revealed that seven mutations examined are localized near both ends of the cupredoxin fold . Met248Ile, Ser258Asn, Phe281Ser, and His284Pro are present in a quinol oxidase-specific (Qox) domain and proximal to low-spin heme b in subunit I and the lost CuA site in subunit II, whereas Ile129Thr, Asn198Thr, and Gln233His are rather scattered in a three-dimensional structure and closer to transmembrane helices of subunit II . Our data suggest that the Qox domain and the CuA end of the cupredoxin fold provide the quinol oxidation site and are involved in electron transfer to the metal centers in subunit I.

FASEB J, 1998 Sep, 12(12), 1191 - 9
Identification and expression of a novel isoform of cAMP response element modulator in the human heart; Muller FU et al.; In end-stage human heart failure, excessive beta-adrenergic stimulation of the cAMP-dependent signaling pathway due to enhanced endogenous catecholamines is hypothesized to contribute to expressional alterations of myocardial regulatory proteins . The cAMP response element modulator (CREM) regulates the transcription of cAMP-responsive genes and might be involved in the regulation of cardiac gene expression . Using the reverse transcription polymerase chain reaction, we identified a novel CREM mRNA, CREM-Ib deltaC-X, in the human heart . Overexpression of CREM-Ib deltaC-X decreased cAMP response element (CRE) -mediated gene transcription in HIT-T15 cells, and this activity was assigned to the part of the sequence encoding putative internally translated proteins . Two of three possible internally translated proteins were immunologically identified in cells overexpressing CREM-Ib deltaC-X tagged with the hemagglutinin epitope of the influenza virus . Both proteins were expressed in bacteria and showed CRE-specific DNA binding, formation of heterodimers with the cAMP response element binding protein (CREB), and inhibition of CREB's binding to the CRE . CREM expression was detected on the mRNA and protein levels in the human heart . We conclude that CREM-Ib deltaC-X generates internally translated repressors of CRE-mediated gene transcription, suggesting the first example for the existence and function of human cardiac CREM.

FASEB J, 1998 Sep, 12(12), 1109 - 23
Expression and activity of prostaglandin endoperoxide synthase-2 in agonist-activated human neutrophils; Pouliot M et al.; Proinflammatory agents were assessed for their capacity to stimulate the expression of the inducible cyclooxygenase isoform (COX-2) in human neutrophils . A number of agents, including PMA, opsonized bacteria and zymosan, LPS, GM-CSF, TNF-alpha, and fMLP, induced COX-2 protein expression through signaling pathways involving transcription and protein synthesis events . Northern blots showed that freshly isolated neutrophils expressed low levels of COX-2 mRNA, which rapidly increased after incubation with inflammatory agents . A characterization of the signal transduction pathways leading to COX-2 protein expression was initiated . In LPS-treated neutrophils, efficient induction of COX-2 required the presence of serum and involved ligand binding to the CD14 surface antigen . The specific inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB 203580, had little effect on the induction of COX-2 expression in neutrophils, in contrast to what had been previously observed with other inflammatory cell types . Depending on the agonist present, ethanol differentially blocked the stimulated expression of COX-2, raising the possibility that phospholipase D activation might take part in the process of COX-2 induction . Major COX-2-derived prostanoids synthesized by inflammatory neutrophils were identified by liquid-chromatography and tandem mass-spectrometry as TXA2 and PGE2 . The agonist-induced synthesis of TXA2 and PGE2 was effectively blocked by cycloheximide and by the specific COX-2 inhibitor NS-398 . These results show that COX-2 can be induced in an active state by different classes of inflammatory mediators in the neutrophil . They support the concept that, in these cells, the COX-2 isoform is preeminent over COX-1 for the stimulated-production of prostanoids, and also suggest that neutrophil COX-2 displays a distinct profile of expression among circulatory cells.

Int J Radiat Biol, 1998 Sep, 74(3), 277 - 86
Conservation of eukaryotic DNA repair mechanisms; Taylor EM et al.; PURPOSE: To discuss the evolutionary conservation of different DNA repair processes . The proteins that carry out base excision repair show a varying degree of structural conservation, but a high level of functional complementation between species, as might be expected for a sequential pathway . In nucleotide excision repair there is a high degree of structural conservation, but few examples of functional complementation because the process involves multiprotein complexes . Repair by homologous recombination involves proteins that are highly conserved structurally . The process of repair of DNA breaks by non-homologous end-joining is conserved in eukaryotes, but the level of sequence identity of several of the proteins is fairly low and some components involved in man do not appear to have sequence homologues in yeast . CONCLUSIONS: All DNA repair processes are highly conserved . The degree of structural and functional conservation varies between the different processes.

J Investig Med, 1998 Aug, 46(6), 275 - 8
Macrophage inflammatory protein-2 predicts acute lung injury in endotoxemia; Kalyanaraman M et al.; BACKGROUND: Proinflammatory mediators that include tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-2 (MIP-2) and anti-inflammatory mediators such as interleukin-10 (IL-10) modulate the immune response to endotoxemia . IL-10 downregulates the production of TNF-alpha and MIP-2 . Acute lung injury may occur secondary to neutrophil chemotaxis mediated by chemokine MIP-2 . We studied the temporal relationship of TNF-alpha, MIP-2, and IL-10 in rat endotoxemia and correlation of MIP-2 concentrations with acute lung injury . METHODS: Ten ventilated rats were randomized to receive an intravenous infusion of 2 mg/kg Escherichia coli lipopolysaccharide (n = 6) or saline placebo (n = 4) . Blood pressure was continuously monitored and arterial blood was obtained for lactate, blood gas, TNF-alpha, IL-10, and MIP-2 measurements at baseline, 2, 4, and 5.5 hours after LPS or saline infusion . RESULTS: Endotoxemia resulted in hypotension, lactic acidemia, and increased alveolar-arterial oxygen gradient (A-a O2 gradient) compared with the placebo group . TNF-alpha, MIP-2, and IL-10 levels were increased 2 hours after endotoxemia . Subsequently, TNF-alpha levels declined while IL-10 and MIP-2 levels remained elevated . Control rats had no significant increase in cytokine production at any time point . MIP-2 concentrations correlated with A-a O2 gradient, an indicator of lung injury (r = 0.56, p < 0.001) . CONCLUSIONS: MIP-2, possibly released by TNF-alpha stimulation of macrophages, is associated with acute lung injury possibly by inducing neutrophil chemotaxis . IL-10 may exert its counter-inflammatory response by inhibiting the release of TNF-alpha in endotoxemia.

Proc Natl Acad Sci U S A, 1998 Sep 15, 95(19), 11471 - 5
One face of a transmembrane helix is crucial in mechanosensitive channel gating; Ou X et al.; MscL is a mechanosensitive channel in bacteria that responds directly to membrane tension by opening a large conductance pore . To determine functionally important residues within this molecule, we have randomly mutagenized mscL, expressed the genes in living bacteria, and screened for gain-of-function mutants with hampered growth . Expression of these genes caused leakage of cytoplasmic solutes on little or no hypo-osmotic stress . In excised patches, the mutant channels gated at membrane tensions that are less than that required for the gating of the wild-type MscL . Hence, the data suggest that the slowed or no-growth phenotype is caused by solute loss because of inappropriate gating of the channel . Most of the mutations mapped to the first transmembrane domain . When this domain is modeled as an alpha-helix, the most severe mutations are substitutions of smaller amino acids (three glycines and one valine) on one facet, suggesting an important role for this structure in MS channel gating.

Proc Natl Acad Sci U S A, 1998 Sep 15, 95(19), 11394 - 9
Presence of a gene encoding choline sulfatase in Sinorhizobium meliloti bet operon: choline-O-sulfate is metabolized into glycine betaine; Osteras M et al.; Glycine betaine is a potent osmoprotectant accumulated by Sinorhizobium meliloti to cope with osmotic stress . The biosynthesis of glycine betaine from choline is encoded by an operon of four genes, betICBA, as determined by sequence and mutant analysis . The betI and betC genes are separated by an intergenic region containing a 130-bp mosaic element that also is present between the betB and betA genes . In addition to the genes encoding a presumed regulatory protein (betI), the betaine aldehyde dehydrogenase (betB), and the choline dehydrogenase (betA) enzymes also found in Escherichia coli, a new gene (betC) was identified as encoding a choline sulfatase catalyzing the conversion of choline-O-sulfate and, at a lower rate, phosphorylcholine, into choline . Choline sulfatase activity was absent from betC but not from betB mutants and was shown to be induced indifferently by choline or choline-O-sulfate as were the other enzymes of the pathway . Unlike what has been shown in other bacteria and plants, choline-O-sulfate is not used as an osmoprotectant per se in S . meliloti, but is metabolized into glycine betaine . S . meliloti also can use this compound as the sole carbon, nitrogen, and sulfur source for growth and that depends on a functional bet locus . In conclusion, choline-O-sulfate and phosphorylcholine, which are found in higher plants and fungi, appear to be substrates for glycine betaine biosynthesis in S . meliloti.

Proc Natl Acad Sci U S A, 1998 Sep 15, 95(19), 11117 - 21
High-affinity binding of hemimethylated oriC by Escherichia coli membranes is mediated by a multiprotein system that includes SeqA and a newly identified factor, SeqB; Shakibai N et al.; The binding of hemimethylated oriC to Escherichia coli membranes has been implicated in the prevention of premature reinitiation at newly replicated chromosomal origins in a reaction that involves the SeqA protein . We describe the resolution of the membrane-associated oriC-binding activity into two fractions, both of which are required for the high-affinity binding of hemimethylated oriC . The active component in one fraction is identified as SeqA . The active component of the second fraction is a previously undescribed protein factor, SeqB . The reconstituted system reproduced the salient characteristics of the membrane-associated binding activity, suggesting that the membrane-associated oriC-binding machinery of E . coli is likely to be a multiprotein system that includes the SeqA and SeqB proteins.

EMBO J, 1998 Sep 15, 17(18), 5477 - 83
McrBs, a modulator peptide for McrBC activity; Panne D et al.; McrBC is a methylation-dependent endonuclease from Escherichia coli K-12 . The enzyme recognizes DNA with modified cytosines preceded by a purine . McrBC restricts DNA that contains at least two methylated recognition sites separated by 40-80 bp . Two gene products, McrBL and McrBs, are produced from the mcrB gene and one, McrC, from the mcrC gene . DNA cleavage in vitro requires McrBL, McrC, GTP and Mg2+ . We found that DNA cleavage was optimal at a ratio of 3-5 McrBL per molecule of McrC, suggesting that formation of a multisubunit complex with several molecules of McrBL is required for cleavage . To understand the role of McrBs, we have purified the protein and analyzed its role in vitro . At the optimal ratio of 3-5 McrBL per molecule of McrC, McrBs acted as an inhibitor of DNA cleavage . Inhibition was due to sequestration of McrC and required the presence of GTP, suggesting that the interaction is GTP dependent . If McrC was in excess, a condition resulting in suboptimal DNA cleavage, addition of McrBs enhanced DNA cleavage, presumably due to sequestration of excess McrC . We suggest that the role of McrBs is to modulate McrBC activity by binding to McrC.

EMBO J, 1998 Sep 15, 17(18), 5449 - 57
Functional analysis of peptide motif for RNA microhelix binding suggests new family of RNA-binding domains; Ribas de Pouplana L et al.; RNA microhelices that recreate the acceptor stems of transfer RNAs are charged with specific amino acids . Here we identify a two-helix pair in alanyl-tRNA synthetase that is required for RNA microhelix binding . A single point mutation at an absolutely conserved residue in this motif selectively disrupts RNA binding without perturbation of the catalytic site . These results, and findings of similar motifs in the proximity of the active sites of other tRNA synthetases, suggest that two-helix pairs are widespread and provide a structural framework important for contacts with bound RNA substrates.

EMBO J, 1998 Sep 15, 17(18), 5438 - 48
L-arginine recognition by yeast arginyl-tRNA synthetase; Cavarelli J et al.; The crystal structure of arginyl-tRNA synthetase (ArgRS) from Saccharomyces cerevisiae, a class I aminoacyl-tRNA synthetase (aaRS), with L-arginine bound to the active site has been solved at 2.75 A resolution and refined to a crystallographic R-factor of 19.7% . ArgRS is composed predominantly of alpha-helices and can be divided into five domains, including the class I-specific active site . The N-terminal domain shows striking similarity to some completely unrelated proteins and defines a module which should participate in specific tRNA recognition . The C-terminal domain, which is the putative anticodon-binding module, displays an all-alpha-helix fold highly similar to that of Escherichia coli methionyl-tRNA synthetase . While ArgRS requires tRNAArg for the first step of the aminoacylation reaction, the results show that its presence is not a prerequisite for L-arginine binding . All H-bond-forming capability of L-arginine is used by the protein for the specific recognition . The guanidinium group forms two salt bridge interactions with two acidic residues, and one H-bond with a tyrosine residue; these three residues are strictly conserved in all ArgRS sequences . This tyrosine is also conserved in other class I aaRS active sites but plays several functional roles . The ArgRS structure allows the definition of a new framework for sequence alignments and subclass definition in class I aaRSs.

EMBO J, 1998 Sep 15, 17(18), 5427 - 37
The deadenylating nuclease (DAN) is involved in poly(A) tail removal during the meiotic maturation of Xenopus oocytes; Korner CG et al.; Exonucleolytic degradation of the poly(A) tail is often the first step in the decay of eukaryotic mRNAs and is also used to silence certain maternal mRNAs translationally during oocyte maturation and early embryonic development . We previously described the purification of a poly(A)-specific 3'-exoribonuclease (deadenylating nuclease, DAN) from mammalian tissue . Here, the isolation and functional characterization of cDNA clones encoding human DAN is reported . Recombinant DAN overexpressed in Escherichia coli has properties similar to those of the authentic protein . The amino acid sequence of DAN shows homology to the RNase D family of 3'-exonucleases . DAN appears to be localized in both the nucleus and the cytoplasm . It is not stably associated with polysomes or ribosomal subunits . Xenopus oocytes contain nuclear and cytoplasmic DAN isoforms, both of which are closely related to the human DAN . Anti-DAN antibody microinjected into oocytes inhibits default deadenylation during progesterone-induced maturation . Ectopic expression of human DAN in enucleated oocytes rescues maturation-specific deadenylation, indicating that amphibian and mammalian DANs are functionally equivalent.

EMBO J, 1998 Sep 15, 17(18), 5255 - 64
Phospholipid-assisted protein folding: phosphatidylethanolamine is required at a late step of the conformational maturation of the polytopic membrane protein lactose permease; Bogdanov M et al.; Previously we presented evidence that phosphatidylethanolamine (PE) acts as a molecular chaperone in the folding of the polytopic membrane protein lactose permease (LacY) of Escherichia coli . Here we provide more definitive evidence supporting the chaperone properties of PE . Membrane insertion of LacY prevents its irreversible aggregation, and PE participates in a late step of conformational maturation . The temporal requirement for PE was demonstrated in vitro using a coupled translation-membrane insertion assay that allowed the separation of membrane insertion from phospholipid-assisted folding . LacY was folded properly, as assessed by recognition with conformation-specific monoclonal antibodies, when synthesized in the presence of PE-containing inside-out membrane vesicles (IOVs) or in the presence of IOVs initially lacking PE but supplemented with PE synthesized in vitro either co- or post-translationally . The presence of IOVs lacking PE and containing anionic phospholipids or no addition of IOVs resulted in misfolded or aggregated LacY, respectively . Therefore, critical folding steps occur after membrane insertion dependent on the interaction of LacY with PE to prevent illicit interactions which lead to misfolding of LacY.

J Cell Biochem, 1998 Oct 1, 71(1), 1 - 10
Dual cytoplasmic and nuclear distribution of the novel arsenite-stimulated human ATPase (hASNA-I); Kurdi-Haidar B et al.; The arsenite-stimulated human ATPase (hASNA-I) protein is a distinct human ATPase whose cDNA was cloned by sequence homology to the Escherichia coli ATPase arsA . Its subcellular localization in human malignant melanoma T289 cells was examined to gain insight into the role of hASNA-I in the physiology of human cells . Immunocytochemical staining using the specific anti-hASNA-I monoclonal antibody 5G8 showed a cytoplasmic, perinuclear, and nucleolar distribution . Subcellular fractionation indicated that the cytoplasmic hASNA-I was soluble and that the perinuclear distribution was due to association with the nuclear membrane rather than with the endoplasmic reticulum . Its presence in the nucleolus was confirmed by showing colocalization with an antibody of known nucleolar specificity . Further immunocytochemical analysis showed that the hASNA-I at the nuclear membrane was associated with invaginations into the nucleus in interphase cells . These results indicate that hASNA-I is a paralogue of the bacterial ArsA protein and suggest that it plays a role in the nucleocytoplasmic transport of a nucleolar component.

J Med Microbiol, 1998 Sep, 47(9), 811 - 20
Molecular probes for the detection of pathogenic fungi in the presence of human tissue; Kappe R et al.; Four primer systems, amplifying fragments of the gene coding for the small ribosomal subunit (18S rRNA) were characterised with pure cultures of 65 medically relevant fungal species plus two mushrooms . A primer cocktail (TR1/CA1-TR2/AF2) amplified 59 of 67 fungal species; the universal fungal primer 1 (UF1) in combination with the eukaryotic primers S3 or EU1 amplified 64 and 65 of 67 fungal species, respectively . The design of an additional primer (RZY1) enabled the amplification of the missing members of the zygomycetes . The primer systems amplified all the medically relevant fungi tested . These included eight Candida spp . and seven other yeast species, 13 dermatophytes, 32 moulds (including six zygomycetes and five dimorphic fungi) and two mushrooms . Eleven controls including DNA from Schistosoma mansoni, Escherichia coli, Mycobacterium tuberculosis and man were not amplified . The oligonucleotide CA hybridised with C . albicans, C . tropicalis and C . parapsilosis; the oligonucleotide TR hybridised with the 13 dermatophytes; the oligonucleotide AF hybridised with Aspergillus fumigatus, A . flavus, A . terreus, A . nidulans, A . versicolor, A . tamarii, A . clavatus, A . fischeri, but not with A . niger or A . versicolor; and the oligonucleotide HC hybridised with three varieties of Histoplasma capsulatum . These oligonucleotides did not hybridise with the other fungi nor the controls . The specificity of the newly designed primer systems was confirmed by selective amplification of fungal DNA from human lung tissue spiked with fungal biomass and from vitrectomy fluid of a patient with candida endophthalmitis.

J Med Microbiol, 1998 Sep, 47(9), 781 - 90
Enteropathogenicity markers in Escherichia coli isolated from infants with acute diarrhoea and healthy controls in Rio de Janeiro, Brazil; Rosa AC et al.; Faeces from urban children < 2 years old with acute diarrhoeal illness and from non-diarrhoeal infants (controls) were examined for Escherichia coli and other enteropathogens . A total of 990 E . coli isolates from 100 patients and 50 controls was tested for enteropathogenic E . coli (EPEC) serotype (O:H), adherence to HEp-2 cells after incubation for 3 and 6 h, fluorescent actin staining (FAS), DNA hybridisation with EAF, eaeA, STh, STp and EAggEC probes and production of heat-labile enterotoxin (LT) and verocytotoxin (VT) with Y1 and Vero cells . EPEC were the most prevalent enteropathogens in patients (32.7%; and 14% in controls) . Enteroinvasive E . coli (EIEC) and Vero cytotoxin-producing E . coli (VTEC) were not detected . The rate of isolation of enterotoxigenic E . coli (ETEC) was identical in both groups . Among the EPEC isolates the prevalent serotypes were O111:H2, O55:NM and O119:H6 . Localised adherence (LA) was found significantly more frequently in isolates from patients (19.6%) than controls (2.1%) . All LA-positive EPEC isolates were FAS+ and eaeA+, but only 75.2% of them hybridised with the EAF probe . Diffusely adhering E . coli (DAEC) and enteroaggregative E . coli (EAggEC) were found with equal frequency in patients and controls . Twenty-seven E . coli isolates were negative for EAF but positive for eaeA and FAS and produced LA in 6-h adherence tests . These EAF-/eaeA+ strains were the only putative enteropathogen identified in seven patients and were not found in controls . The ability of these strains to elicit ultrastructural cell alterations and cell-signalling events was evaluated in Caco-2 cells (human colon carcinoma cell line) by the gentamicin invasion assay and by transmission electron microscopy . The numbers of intracellular bacteria in cell invasion tests varied from 0.4% to 1.6% of the cell-associated bacteria after a 6-h incubation period . Tyrosine phosphorylation of host cell proteins was assessed in HEp-2 cells by immunofluorescence microscopy and all strains gave positive results . EAF-/eaeA+ E . coli strains express most of the virulence properties found among true EPEC strains and can be a relevant cause of infant diarrhoea in developing countries.

Korean J Intern Med, 1998 Jul, 13(2), 95 - 8
Detection of antibodies against DNA polymerase of hepatitis B virus in HBsAg-positive sera using ELISA; Rui LX et al.; OBJECTIVES: DNA polymerase (pol) of Hepatitis B virus (HBV) includes 3 different domains such as terminal protein (TP), reverse transcriptase (RT) and RNase H . Humoral immune responses to each of these proteins have not been well documented previously, although antibody to pol was detected in serum of patients with chronic hepatitis B . We have constructed TP (amino acids 1-182), RT (amino acids 346-685) and RNase H (amino acids 690-832) . METHODS: By ELISA using each protein expressed in E . coli as antigens, the corresponding antibodies were tested in serum from 40 patients with type B viral chronic liver diseases . (20 HBeAg-positive and 20 HBeAg-negative) . As negative controls, sera from 3 healthy young men were used . With the mean values of the OD, which were tested 4 times per each test sample and 3 times per each control sample, we considered to be positive if the mean OD of each test sample is 2-fold or higher than that of controls . RESULTS: Five of 40 sera (12.5%) contained one or two different antibodies detectable by this method: 4 of 20 HbeAg-positive sera (20%) and 1 of 20 HbeAg-negative sera (5%) . Anti-TP, anti-RT and anti-RNase H antibodies were detected in 2.5% (1/40), 10% (4/40) and 7.5% (3/40), respectively . Among 4/20 HbeAg-positive ELISA-positive sera, anti-TP, anti-RT and anti-RNase H were positive in 5% (1/20), 20% (4/20) and 10% (2/20), respectively, while 1 HBeAg-negative ELISA-positive sera were positive only for anti-RNase H . CONCLUSIONS: These results suggest that the corresponding antibody responses to individual recombinant peptides derived from 3 domains of DNA polymerase may tend to be detected more frequently in HBeAg-positive sera than in HBeAg-negative sera from various patients with type B viral chronic liver diseases.

J Radiat Res (Tokyo), 1998 Jun, 39(2), 137 - 44
3'-blocking damage of DNA as a mutagenic lesion caused by hydrogen peroxide in Escherichia coli; Takemoto T et al.; Ionizing radiation and hydrogen peroxide (H2O2) produce many types of oxidative DNA damage such as strand breaks, apurinic/apyrimidinic (AP) sites, base modifications and 3'-blocking damage such as 3'-phosphoglycolated and 3'-phosphorylated termini . AP sites and 3'-blocking damage are repairable by exonuclease III and endonuclease IV in Escherichia coli . XthA-nfo double mutants of E . coli, which are deficient in exonuclease III and endonuclease IV, were highly sensitive to lethal and mutagenic effects of H2O2, compared with the wild-type strains . The pNT180 and pNT186 plasmids containing wild-type nfo and mutant nfo-186 gene, respectively, were introduced into the xthA-nfo mutant . The nfo-186 gene product, Nfo186, retained normal AP endonuclease activity but could not remove 3'-blocking damage from DNA . The pNT180 corrected the sensitivity of the xthA-nfo mutant to lethal and mutagenic effects of H2O2 . On the other hand, the pNT186 did not have any complementation effects . From these results it was concluded that 3'-blocking damage rather than an AP site is the primary lesion responsible for both lethal and mutagenic effects of H2O2.

Can J Public Health, 1998 Jul-Aug, 89(4), 253 - 7
A descriptive study of verocytotoxigenic Escherichia coli (VTEC) cases reported in Ontario, 1990-1994; Michel P et al.; Characteristics of VTEC cases identified through routine surveillance in Ontario between 1990 and 1994 are described . Information was extracted from the Reportable Disease Information System (RDIS) of Ontario and was evaluated for its completeness and internal validity . A total of 2,441 VTEC cases were identified for the five-year study period corresponding to an average annual rate of 4.8 cases per 100,000 . Sixteen deaths were recorded . Bloody diarrhea was reported for 546 patients (40%) and was the most frequently reported symptom . For most cases, the home was recorded as the likely risk setting (36%) . Food was incriminated as the source of infection for more than 36% of cases . Nine (69%) of the thirteen data fields compulsory for transmission to the Ontario Ministry of Health had less than 10% of combined missing and unspecified values . Fields describing risk factors had greater than 56% of entries missing or unspecified.

Hum Mol Genet, 1998, 7(10), 1573 - 9
Nuclear genes of human complex I of the mitochondrial electron transport chain: state of the art; Smeitink JA et al.; The mitochondrial electron transport chain (mtETC) consists of four multi-subunit enzyme complexes . Complex I or NADH:ubiquinone oxidoreductase, the largest mtETC multisubunit complex, consists of approximately 41 subunits . Seven of these subunits are encoded by the mitochondrial genome, the remainder by the nuclear genome . Among the mitochondriocytopathies, complex I deficiencies are encountered frequently . Although some complex I deficiencies have been associated with mitochondrial DNA mutations, the genetic defect has not been elucidated in the majority of complex I-deficient patients . It is expected that many of these patients have mutations in the nuclear-encoded subunits of this complex, so vital for cellular energy production . After a brief summary of the current knowledge of complex I from cow, bacteria and fungi, this review presents the state of the art of the knowledge of the human nuclear-encoded complex I genes which, in the last 18 months, has made enormous progress . At present, the complete gene structure of four subunits and the cDNA structure of 18 of the 34 complex I nuclear-encoded subunits are known . Mapping of these subunits shows a random distribution over the chromosomes . The chromosomal localization is known for 14 complex I genes . Recently, the first mutation, a 5 bp duplication in the 18 kDa (AQDQ) subunit, has been reported . We expect that within 1 year all human nuclear-encoded complex I subunits will be cloned . Mutational analysis of these subunits is warranted in complex I-deficient patients and will not only be important for genetic counselling but will also extend the knowledge regarding the functional properties of the individual human complex I subunits.

Biochem Biophys Res Commun, 1998 Sep 8, 250(1), 154 - 60
Characterization of phospholipase A2 (PLA2) from Taiwan Cobra: isoenzymes and their site-directed mutants; Pan FM et al.; Extracellular and secretory phospholipase A2 (PLA2), a class of phospholipid digesting enzyme, is widely distributed in animal venoms of reptiles and insects . Two cDNAs encoding PLA2 isoenzymes from Taiwan Cobra (Naja naja atra) were cloned into pQE-30 plasmid vector and expressed in Escherichia coli . The recombinant products were subjected to refolding using sulfonation under reduction/oxidation conditions with glutathione and enterokinase removal of His-tag, resulting in the active recombinant PLA2 with the same molecular masses of native enzymes as determined by mass spectrometry . The recombinant PLA2 was also shown by circular dichroism to possess a secondary structure similar to native PLA2 . The enzymatic activity of the major isoenzyme (PLA2-1) is higher than the other minor isoenzyme (PLA2-2), which shows two amino acid difference from PLA2-1 . Site-directed mutagenesis was used to probe the structure/function relationship of two highly conserved residues among all reported PLA2, i.e., His-47 and Asp-93 . Replacement of His-47 residue by either Ala or Arg resulted in the complete loss of activity . Similarly, the mutant Asp-93 --> Asn (D93N) also retained little activity . These results suggest that both His-47 and Asp-93 are essential for the catalytic activity of PLA2 . Computer graphic study, based on homology modelling, highlights the differences between native PLA2 isoenzymes and their site-directed mutants, which may account for the differences in the observed biological activity .

Biochem Biophys Res Commun, 1998 Sep 8, 250(1), 115 - 8
Hsc62, a new DnaK homologue of Escherichia coli; Yoshimune K et al.; We have cloned and expressed the ORF o170#1 of Escherichia coli, which encodes a 62-kDa protein sharing 33% homology in primary structure with DnaK and Hsc66, Hsp70 homologues of E . coli . The purified gene product, which we named Hsc62, clearly showed ATPase activity and was bound to a gelatin-agarose gel, from which it was specifically eluted with ATP magnesium salt . Thus, Hsc62 is similar to DnaK in this respect and probably functions as a molecular chaperon in E . coli . However, Hsc62 differs markedly from DnaK and also from Hsc66 in response to temperature: the optimum temperature for ATPase activity was increased stepwise in the order of Hsc62, Hsc66, and DnaK . Hsc66 is activated by DnaJ of E . coli in the same manner as DnaK, the natural partner protein of DnaJ . However, Hsc62 is distinct from the others: the ATPase activity of Hsc62 was not elevated by DnaJ .

Biochem Biophys Res Commun, 1998 Sep 8, 250(1), 32 - 5
LonR9 carrying a single Glu614 to Lys mutation inhibits the ATP-dependent protease La (Lon) by forming mixed oligomeric complexes; Oh JY et al.; An unusual lon mutation (called lonR9) is dominant over the wild-type gene, which encodes the ATP-dependent protease La (Lon) in Escherichia coli, when present in multicopy plasmids . Here, we cloned and sequenced lonR9, and showed that the mutant gene carries a single point mutation in its open reading frame, which leads to replacement of Glu614 by Lys . The LonR9 protein and its poly-His-tagged form were purified to apparent homogeneity . Both of the purified proteins were capable of inhibiting the ATP-dependent proteolysis and the protein-activated ATP hydrolysis by protease La . Furthermore, the His-tagged LonR9 protein was found to form mixed oligomeric complexes with protease La, upon analysis by chromatography on a metal-chelating column . These results suggest that the phenotypic dominance of the lonR9 mutant is due to the formation of mixed oligomeric complexes between LonR9 and protease La, in which the defective components prevent the function of the wild-type subunits .

Biochem Biophys Res Commun, 1998 Sep 8, 250(1), 5 - 11
Neuropeptide specificity and inhibition of recombinant isoforms of the endopeptidase 3.4.24.16 family: comparison with the related recombinant endopeptidase 3.4.24.15; Rioli V et al.; Endopeptidase EC 3.4.24.16 (EP24.16c, neurolysin) and thimet oligopeptidase EC 3.4.24.15 are close related members of a large family of metalloproteases . Besides their cytosolic and membrane bound form, endopeptidase EC 3.4.24.16 appears to be present in the inner membrane of the mitochondria (EP24.16m) . We have overexpressed two porcine EP24.16 isoforms in E . coli and purified the recombinant proteins to homogeneity . We show here that these peptidases hydrolyse a series of neuropeptides with similar rates and at sites reminiscent of those elicited by classically purified human brain EP24.16c . All neuropeptides, except neurotensin, were similarly cleaved by recombinant endopeptidase 3.4.24.15 (EP24.15, thimet oligopeptidase), another zinc-containing metalloenzyme structurally related to EP24.16 . These two EP24.16 isoforms were drastically inhibited by Pro-Ile and dithiothreitol and remained unaffected by a specific carboalkyl inhibitor (CFP-AAY-pAb) directed toward the related EP24.15 . The present purification procedure of EP24.16 should allow to establish, by mutagenesis analysis, the mechanistic properties of the enzyme .

Plasmid, 1998 Sep, 40(2), 150 - 7
Effect of integration host factor of RNA II synthesis in replication of plasmid containing orip 15A; Hiszczynska-Sawicka E et al.; The synthesis rates of the replication control RNAs of plasmid orip15A . RNA I, an inhibitor of replication, and RNA II, the primer, have been determined using lacZ fusion plasmids, hybridization assay, and reverse transcription polymerase chain reaction (RT-PCR) in Escherichia coli integration host factor-positive (IHF+) and -negative (IHF-) strains containing pACYC184 plasmid (orip15A) . In the absence of IHF (E . coli IHF-), expression of the lacZ gene from the PRNAII promoter increased by a factor of 4 compared with the E . coli wild type (IHF+) . Also, the increase in expression was more pronounced when the IHF protein was mutated in the ihfB gene than in the ihfA gene . For the PRNAII promoter of oripMB1 (pBR322), no significant differences were found in expression of the lacZ gene in he E . coli strains examined . The level of beta-galactosidase expression from the PRNA promoter of orip 15A shows that the absence of functional IHF in the transformed strains has no effect on expression of the lacZ gene . The synthesis RNA II:RNA I ratio obtained in hybridization assays was 2.4 for E . coli IHF+ and 4.4 for E . coli IHF- . Densitometric analysis of RT-PCR products indicates that the relative levels of RNA I in E . coli IHF+ and IHF-, are equal, but the relative level of RNA II in E . coli IHF is about four times higher than in E . coli IHF+ . These results indicate that the IHF protein inhibits transcription from the PRNAII promoter of orip15A plasmid.

Plasmid, 1998 Sep, 40(2), 140 - 9
Functional domains of Rts1 and P1 RepA proteins for initiation of replication; Li YF et al.; Rts1 RepA and P1 RepA are trans-acting proteins essential for initiation of replication of Rts1 and P1 plasmids, respectively . We recently found that P1 RepA bound in vitro to the Rts1 replication origin as strongly as Rts1 RepA and activated the origin in vivo . However, the ori activation was quite inefficient . This study shows that by replacing a small region of P1 RepA with the corresponding region of Rts1 RepA, the efficiency of Rts1 ori activation increased markedly . Interestingly, the same subregion of P1 RepA was found to be important for in vivo activation of the P1 origin . Thus, a region essential for efficient activation of the replication origin was assigned to the P1 RepA molecule as well as to the Rts1 RepA molecule . The region was distinct from a domain necessary for in vitro binding to the origin, although both regions were required for in vivo activation of the respective origin.

Plasmid, 1998 Sep, 40(2), 91 - 9
The central lysine in the P-loop motif of the Escherichia coli DnaA protein is essential for initiating DNA replication from the chromosomal origin, oriC, and the F factor origin, oriS, but is dispensable for initiation from the P1 plasmid origin, oriR; Skovgaard O et al.; The Escherichia coli DnaA protein is essential for initiation of DNA replication from the chromosomal origin, oriC, and from certain plasmid origins such as oriR of P1, oriS of F, and ori of pSCS101 . The DnaA protein binds ATP with high affinity and contains a P-loop motif assumed to be the binding site . Three mutations in the E . coli dnaA gene were constructed by oligonucleotide-directed mutagenesis that changed amino acids in the P-loop . A DnaA protein, K178T, in which the central lysine was changed to the smaller amino acid threonine, was able to initiate DNA replication from P1 oriR, but was unable to initiate replication from E . coli oriC or F oriS in vivo . Mutant and wild-type DnaA proteins were overexpressed, partially purified, and tested for replication activity in vitro . The K178T DnaA protein could initiate replication from oriR, although with a decreased activity compared to the wild-type DnaA protein . No replication activity was detected for this mutant protein from oriC . The different responses of the oriR and oriC replicons to the K178T DnaA protein indicate that the role of DnaA is different in the two systems.

Methods, 1998 Jul, 15(3), 255 - 63
MxA GTPase: oligomerization and GTP-dependent interaction with viral RNP target structures; Kochs G et al.; MxA protein is an interferon-induced GTPase of human cells that inhibits the multiplication of several RNA viruses, including influenza viruses and bunyaviruses . Studies on MxA transgenic mice have shown that MxA is a powerful antiviral agent in vivo . It has been suggested that this cellular protein also protects humans from viral disease, but the mechanism(s) by which MxA exerts its antiviral action is still poorly understood . Using an in vitro cosedimentation assay, we now demonstrate that MxA tightly interacts with components of the ribonucleoprotein complex of Thogoto virus, an influenza-like virus transmitted by ticks . This assay demonstrates for the first time a physical interaction between MxA GTPase and a viral target structure . It is based on three elements, namely, highly active MxA GTPases as effector molecules, viral ribonucleoprotein particles as viral targets, and GTPgammaS as a stabilizing factor . Furthermore, using a simple nuclear translocation assay, we show that human MxA protein forms oligomers in vivo . This assay provides a stringent test for tight association of partner molecules in intact mammalian cells . It not only will be useful for studying physical interactions of MxA with partner molecules, but may also be applicable to other studies on protein-protein interactions in living cells .

Methods, 1998 Jul, 15(3), 225 - 32
Characterization of viral double-stranded RNA-binding proteins; Jacobs BL et al.; Several viruses have been shown to code for proteins that specifically bind to double-stranded RNA or RNA with large amounts of secondary structure . These proteins have been implicated in providing interferon resistance to viruses and in inhibiting induction of apoptosis by viruses, and have been suggested to be involved in regulation of viral and cellular protein synthesis in infected cells . This article describes methods for detecting and analyzing viral double-stranded RNA-binding proteins .

Methods, 1998 Jul, 15(3), 207 - 23
Using genetic means to dissect homologous and heterologous protein-protein interactions of PKR, the interferon-induced protein kinase; Tan SL et al.; The interferon-induced protein kinase, PKR, is a pivotal component of interferon (IFN)-induced cellular antiviral and antiproliferative response . The identification and characterization of proteins, of both viral and cellular origins, that interact with PKR have proven to be a valuable probe for unraveling the cellular regulation and function of PKR . Several studies have demonstrated that PKR forms dimers and that dimerization is likely to be required for activation and/or catalytic function . It is therefore important to elucidate the mechanism of PKR dimer formation and the role of PKR effectors in modulating kinase dimerization . Herein we describe the use of the two genetic approaches, the lambda repressor fusion and the yeast two-hybrid systems, to detect and analyze homo- and heterotypic interactions with PKR . We also describe several biochemical methodologies commonly used in our laboratory to validate the genetic results . Although the examples in this article focus on PKR, the techniques can easily be adapted to investigate protein-protein associations in a variety of experimental systems . Finally, given the important role of PKR as a mediator of IFN-induced antiviral and antiproliferative effects, these studies may provide clues to the development of reagents that target PKR to enhance the therapeutic use of IFN in the treatment of disease .

J Mol Biol, 1998 Sep 18, 282(2), 275 - 85
SSB protein controls RecBCD enzyme nuclease activity during unwinding: a new role for looped intermediates; Anderson DG et al.; The RecBCD enzyme of Escherichia coli initiates homologous recombination by unwinding and simultaneously degrading DNA from a double-stranded DNA end . Single-stranded DNA loops are intermediates of this unwinding process . Here we show that SSB protein reduces the level of DNA degradation by RecBCD enzyme during unwinding, by binding to these ssDNA intermediates . Prior to interaction with the recombination hot spot chi, RecBCD enzyme has both 3'-->5' exonuclease and a weaker 5'-->3' exonuclease activity . We show that degradation of the 5'-terminal strand at the entry site is much more extensive in the absence of SSB protein . After interaction with chi, the level of 5'-->3' exonuclease activity is increased; as expected, degradation of the 5'-strand is also elevated in the absence of SSB protein . Furthermore, we show that, in the absence of SSB protein, the RecBCD enzyme is inhibited by the ssDNA products of unwinding; SSB protein alleviates this inhibition . These results provide insight into the organization of helicase and nuclease domains within the RecBCD enzyme, and also suggest a new level at which the nuclease activity of RecBCD enzyme is controlled . Hence, they offer new insight into the role of SSB protein in the initiation phase of recombination .

J Mol Biol, 1998 Sep 18, 282(2), 255 - 64
ErmE methyltransferase recognition elements in RNA substrates; Vester B et al.; Dimethylation by Erm methyltransferases at the N-6 position of adenine 2058 (A2058, Escherichia coli numbering) in domain V of bacterial 23 S rRNA confers resistance to the macrolide-lincosamide-streptogramin B (MLS) group of antibiotics . The ErmE methyltransferase from Saccharopolyspora erythraea methylates a 625 nucleotide transcript of domain V as efficiently as it methylates intact 23 S rRNA . By progressively truncating domain V, the motif required for specific recognition by the enzyme has been localized to a helix and single-stranded region adjacent to A2058 . The smallest RNA transcript that shows methyl-accepting activity is a 27-nucleotide stem-loop, corresponding to the 23 S rRNA sequences 2048 to 2063 and 2610 to 2620 (helix 73), with A2058 situated within the hairpin loop . Methylation of A2058 in the truncated RNAs is optimal in the absence of magnesium, and the efficiency of methylation is halved by the presence of 2 to 3 mM magnesium . Magnesium serves to stabilize a conformation in the truncated RNA that prevents efficient methylation . This contrasts to the intact domain V RNA, where 2 mM magnesium ions support a conformation at A2058 that is most readily recognized by ErmE . Methylation of domain V RNA is generally far less susceptible to ionic conditions than the truncated RNAs . The effects of monovalent cations on the methylation of truncated transcripts suggest that RNA structures outside helix 73 support the ErmE interaction . However, interaction with these structures is not essential for specific ErmE recognition of A2058 .

Arch Biochem Biophys, 1998 Sep 15, 357(2), 240 - 8
Identification of residues 286 and 289 as critical for conferring substrate specificity of human CYP2C9 for diclofenac and ibuprofen; Klose TS et al.; Specificity of human CYP2C9 for two substrates, diclofenac and ibuprofen, was studied using chimeras and site-directed mutants of CYP2C9 and the highly related CYP2C19 expressed in Escherichia coli . Data were correlated with the presence of putative substrate recognition sites (SRS) . A CYP2C19 chimera containing residues 228-340 (SRS 3 and 4) of 2C9 conferred both diclofenac hydroxylation and 2- and 3-hydroxylation of ibuprofen . The regiospecificity of this construct for metabolism of ibuprofen differed from that of CYP2C9 by favoring 2-hydroxylation over 3-hydroxylation . A CYP2C9 construct containing residues 228-340 of CYP2C19 lacked both diclofenac and ibuprofen hydroxylase activities . When residues 228-282 (containing SRS 3) of CYP2C9 were replaced by those of CYP2C19, the chimera retained appreciable activity for diclofenac and ibuprofen, and tolbutamide activity was inhibited by a specific CYP2C9 inhibitor, sulfaphenazole . This suggested that SRS 3 is not important in conferring specificity . CYP2C9 and CYP2C19 differ in five residues within the region 283-340 (within SRS 4) . Mutations to analyze SRS 4 were made on a CYP2C19 chimera containing residues 228-282 of CYP2C9 . A single I289N mutation conferred a dramatic increase in diclofenac hydroxylation and a small increase in ibuprofen 2-hydroxylation . A second mutation (N286S and I289N) increased diclofenac hydroxylation and conferred a dramatic increase in ibuprofen 2-hydroxylation . A V288E mutation did not increase activity toward either substrate and decreased activity toward the two substrates in combination with the I289N or the N286S, I289N mutants . Therefore residues 286 and 289 of CYP2C9 are important in conferring specificity for diclofenac and ibuprofen.

Arch Biochem Biophys, 1998 Sep 15, 357(2), 225 - 30
Suppression of copper-induced cellular damage by copper sequestration with S100b protein; Shiraishi N et al.; We previously reported that S100b protein (homodimer of S100 beta subunit) can bind copper ions with a submicromolar dissociation constant (T . Nishikawa et al., J . Biol . Chem . 272, 23037-23041, 1997) . In this study, a question was addressed as to whether this protein can sequester copper ions in an in vivo situation . Escherichia coli cells that had been rendered able to produce a fusion protein of rat S100 beta subunit with glutathione S-transferase displayed a marked resistance to cellular damage induced by copper alone or its combination with H2O2, compared with control cells expressing the transferase moiety only . A study by gel chromatography showed that about half of the expressed S100 beta fusion protein in the cytosol of copper-treated cells was eluted in the void volume fraction (molecular mass > 200 kDa), which contained most of the copper incorporated . The S100 beta fusion protein purified from the void volume fraction was found to contain 82% of the total copper in the fraction, while in a parallel experiment with the control cells, the glutathione S-transferase eluted in the void volume fraction contained only 18% of the total copper . Thus, it is clear that extraneously expressed S100b protein can acts as a "copper sink," thereby protecting E . coli cells from copper-induced cellular damage.

Biochim Biophys Acta, 1998 Aug 14, 1373(1), 237 - 51
Isolation and sequence of omcA, a gene encoding a decaheme outer membrane cytochrome c of Shewanella putrefaciens MR-1, and detection of omcA homologs in other strains of S . putrefaciens; Myers JM et al.; The sequence of the omcA gene, which encodes a decaheme cytochrome c that is localized to the outer membrane (OM) of Shewanella putrefaciens MR-1, was determined . The 2202 bp nucleotide sequence of omcA encodes for 734 amino acids with a predicted molecular protein mass of 78.6 kDa . Comparison with the amino-terminal sequence of the mature protein suggests the presence of a hydrophobic leader sequence which is cleaved during translocation of the protein to the OM . This leader sequence has a lipoprotein consensus sequence for signal peptidase II at the cleavage site . The predicted mature protein is comprised of 708 amino acids with a predicted molecular mass of 75.8 kDa, but the addition of ten covalently attached heme c groups and covalent lipid modification to the amino-terminal cysteine increases the predicted mass to 82.7 kDa . This is consistent with its apparent mass of 83 kDa in SDS-PAGE gels . The predicted amino acid sequence for the OmcA protein shows no significant homology to known proteins . A RNA of approx . 2300 bases that hybridizes to the omcA gene was detected in anaerobically grown MR-1 cells . The size of this transcript is similar to the coding region of the omcA gene, suggesting that it is not part of a multicistronic operon . Similar to MR-1, four other strains of S . putrefaciens were all found to localize a majority of their membrane-bound cytochromes to the OM when grown under anaerobic conditions, and all contained an OM cytochrome of similar size to OmcA . In two of these strains, MR-4 and MR-8, a homolog of omcA was identified by RT-PCR and Southern blotting using primers and probes specific for omcA of MR-1 . Western blot analysis using a polyclonal antibody to OmcA was similarly positive in strains MR-4 and MR-8 . Partial nucleotide sequence analysis of these homologs demonstrated 74-77% predicted amino acid homology with OmcA of MR-1 . In contrast, strains MR-30 and MR-42 tested negative for omcA homologs by Southern and Northern blots, RT-PCR, and Western blots.

Genes Cells, 1998 Jun, 3(6), 331 - 45
Regulatory mechanisms in expression of the traY-I operon of sex factor plasmid R100: involvement of traJ and traY gene products; Taki K et al.; BACKGROUND: The plasmid R100 encodes tra genes essential for conjugal DNA transfer in Escherichia coli . Genetic evidence suggests that the traJ gene encodes a positive regulator for the traY-I operon, which includes almost all the tra genes located downstream of traJ . The molecular mechanism of regulation by TraJ, however, is not yet understood . traY is the most proximal gene in the traY-I operon . TraY promotes DNA transfer by binding to a site, sbyA, near the origin of transfer . TraY is suggested to have another role in regulation of the traY-I operon, since it binds to two other sites, named sbyB and sbyC, located in the region preceding traY-I . RESULTS: Using a traY-lacZ fusion gene, we showed that the traY-I operon was expressed only in the presence of traJ . The TraJ-dependent expression of traY-I required the E . coli arcA gene, which encodes a host factor required for conjugation . TraJ-dependent transcription occurred from a promoter (named pY) located upstream of traY-I . The isolated TraJ protein was found to bind to a dyad symmetry sequence, named sbj (specific binding site of TraJ), which existed in the intergenic region between traJ and traY-I . We also demonstrated that TraY repressed the TraJ-dependent expression of traY-I at the TraY binding sites, sbyB and sbyC, which overlapped with pY . CONCLUSIONS: TraJ is a protein which binds to the sbj site in the region upstream of the promoter pY and positively regulates expression of the traY-I operon in the presence of the E . coli arcA gene . Since sbj is located 93bp upstream of pY in the intergenic region between traJ and traY-I, TraJ presumably contacts with a transcription apparatus to promote transcription from pY . TraY, which is known to activate the initiation of conjugal DNA transfer, has a new role in the transcriptional autoregulation of traY-I expression . At levels which are sufficient to initiate conjugal DNA transfer, TraY represses traY-I transcription in the presence of TraJ.

Mol Cell, 1998 Aug, 2(2), 191 - 9
The mutagenesis proteins UmuD' and UmuC prevent lethal frameshifts while increasing base substitution mutations; Reuven NB et al.; Error-prone DNA repair consists of replicative filling-in of DNA gaps carrying lesions . We have reconstituted E . coli SOS error-prone repair using purified DNA polymerase III holoenzyme, SSB, RecA, UmuD', a UmuC fusion protein, and a gap lesion plasmid . In the absence of UmuDC, or without SOS induction, replication skips over the lesion, forming mostly one-nucleotide deletions . These cause translational frameshifts that usually inactivate genes . UmuD' and UmuC, in the presence of RecA and SSB, stimulate translesion replication and change its mutagenic specificity such that deletions are prevented and base substitutions are increased . This results in mutagenic but nondetrimental gap repair and provides an effective mechanism for generating genetic variation in bacteria adapting to environmental stress.

J Virol, 1998 Oct, 72(10), 8158 - 65
Functional analysis of the human cytomegalovirus US28 gene by insertion mutagenesis with the green fluorescent protein gene; Vieira J et al.; The protein encoded by the US28 gene of human cytomegalovirus (HCMV) has homology to G protein-coupled receptors (GCR) . Previous studies demonstrated that recombinant US28 protein can bind the beta class of chemokines (K . Neote, D . DiGregorio, J . Y . Mak, R . Horuk, and T . J . Schall, Cell 72:415-425, 1993) and induce a rise in intracellular calcium after the binding of chemokines (J . L . Gao and P . M . Murphy, J . Biol . Chem . 269:28539-28542, 1994) . In order to investigate the function of the US28 protein in virus-infected cells, a recombinant HCMV (HV5.8) was constructed, with the US28 open reading frame disrupted by the insertion of the Escherichia coli gpt gene and the gene for the green fluorescent protein . The US28 gene is not required for growth in human fibroblasts (HF) . HF infected with wild-type HCMV bound RANTES at 24 h postinfection and demonstrated an intracellular calcium flux induced by RANTES . In cells infected with HV5.8, RANTES did not bind or induce a calcium flux, demonstrating that US28 is responsible for the beta-chemokine binding and induced calcium signaling in HCMV-infected cells . The ability of the US28 gene to bind chemokines was shown to cause a significant reduction in the concentration of RANTES in the medium of infected cells . Northern analysis of RNA from infected cells showed that US28 is an early gene, while US27 (another GCR) is a late gene.

J Virol, 1998 Oct, 72(10), 8089 - 97
Avian hepatitis B virus infection is initiated by the interaction of a distinct pre-S subdomain with the cellular receptor gp180; Urban S et al.; Functionally relevant hepadnavirus-cell surface interactions were investigated with the duck hepatitis B virus (DHBV) animal model by using an in vitro infection competition assay . Recombinant DHBV pre-S polypeptides, produced in Escherichia coli, were shown to inhibit DHBV infection in a dose-dependent manner, indicating that monomeric pre-S chains were capable of interfering with virus-receptor interaction . Particle-associated pre-S was, however, 30-fold more active, suggesting that cooperative interactions enhance particle binding . An 85-amino-acid pre-S sequence, spanning about half of the DHBV pre-S chain, was characterized by deletion analysis as essential for maximal inhibition . Pre-S polypeptides from heron hepatitis B virus (HHBV) competed DHBV infection equally well despite a 50% difference in amino acid sequence and a much-reduced infectivity of HHBV for duck hepatocytes . These observations are taken to indicate (i) that the functionality of the DHBV pre-S subdomain, which interacts with the cellular receptor, is determined predominantly by a defined three-dimensional structure rather than by primary sequence elements; (ii) that cellular uptake of hepadnaviruses is a multistep process involving more than a single cellular receptor component; and (iii) that gp180, a cellular receptor candidate unable to discriminate between DHBV and HHBV, is a common component of the cellular receptor complex for avian hepadnaviruses.

J Virol, 1998 Oct, 72(10), 7830 - 9
Serp2, an inhibitor of the interleukin-1beta-converting enzyme, is critical in the pathobiology of myxoma virus; Messud-Petit F et al.; Recently, myxoma virus was shown to encode an additional member of the serpin superfamily . The viral gene, called serp2, was cloned, and the Serp2 protein was shown to specifically bind to interleukin-1beta (IL-1beta)-converting enzyme (ICE), thus inhibiting the cleavage of pro-IL-1beta by the protease (F . Petit, S . Bertagnoli, J . Gelfi, F . Fassy, C . Boucraut-Baralon, and A . Milon, J . Virol . 70:5860-5866, 1996) . Here, we address the role of Serp2 in the development of myxomatosis, a lethal infectious disease of the European rabbit . A Serp2 mutant myxoma virus was constructed by disruption of the single-copy serp2 gene and insertion of the Escherichia coli gpt gene serving as the selectable marker . A revertant virus was obtained by replacing the E . coli gpt gene by the intact serp2 open reading frame . The Serp2(-) mutant virus replicated with wild-type kinetics both in rabbit fibroblasts and a rabbit CD4(+) T-cell line (RL5) . Moderate reduction of cell surface levels of major histocompatibility complex I was observed after infection with wild-type or Serp2(-) mutant myxoma virus, and both produced white pocks on the chorioallantoic membrane of the chick embryo . After the infection of European rabbits, the Serp2(-) mutant virus proved to be highly attenuated compared to wild-type myxoma virus, as demonstrated by the clinical course of myxomatosis and the survival rates of infected animals . Pathohistological examinations revealed that infection with wild-type myxoma virus resulted in a blockade of the inflammatory response at the vascular level . In contrast, rapid inflammatory reactions occurred upon infection with the Serp2(-) mutant virus . Furthermore, lymphocytes in lymph nodes derived from animals inoculated with Serp2 mutant virus were shown to rapidly undergo apoptosis . We postulate that the virulence of myxoma virus in the European rabbit can be partially attributed to an impairment of host inflammatory processes and to the prevention of apoptosis in lymphocytes . The weakening of host defense is directly linked to serp2 gene function and is likely to involve the inhibition of IL-1beta-converting-enzyme-dependent pathways.

J Biol Chem, 1998 Sep 18, 273(38), 24906 - 11
Requirements for and regulation of origin opening of plasmid P1; Park K et al.; Origin opening is essential for the initiation of DNA replication in the theta mode and requires binding of initiator proteins . Using reactivity to KMnO4 in vivo as an assay, we find that, like initiation, origin opening of the Escherichia coli plasmid P1 requires the host initiators DnaA and HU and the plasmid-encoded initiator RepA . The ability to detect opening at the P1ori in vivo allowed us to study this activity at various copy numbers in chimeric replicons . The opening was prevented when the P1ori was cloned in high copy vectors or when excess RepA binding sites (iterons) were provided in trans . However, when RepA supply was also increased, the opening was efficient . A further increase in RepA prevented opening . Replication of an incoming P1 under these conditions correlated with opening . These results demonstrate that initiation is possible even at abnormally high origin concentrations and that oversupply of RepA, relative to iterons, can prevent replication by blocking origin opening . It appears that plasmid overreplication can be prevented either by limiting RepA or by accumulating RepA at a rate higher than that of the origin.

J Biol Chem, 1998 Sep 18, 273(38), 24744 - 53
The cation-binding domain from the alpha subunit of integrin alpha5 beta1 is a minimal domain for fibronectin recognition; Baneres JL et al.; The cation-binding domain from the alpha subunit of human integrin alpha5beta1 was produced as a recombinant protein, alpha5-(229-448) . This protein displays a well defined fold with a content of 30-35% alpha-helix and 20-25% beta-strand, based on circular dichroism . The binding of Ca2+ or Mg2+ to alpha5-(229-448) results in a biphasic conformational rearrangement consistent with the occurrence of two classes of cation-binding sites differing by their affinities . The two classes of sites are located in two conformationally independent lobes, as established by a parallel study of two recombinant half-domains (N- and C-terminal) that also adopt stable folds . Upon saturation with divalent cations, alpha5-(229-448) binds an Arg-Gly-Asp (RGD)-containing fibronectin ligand to form a 1:1 complex . Complex formation is associated with a specific conformational adaptation of the ligand, suggesting an induced fit mechanism . In contrast, neither of the half-domains is competent for ligand binding . The alpha5-(229-448)-fibronectin complex is dissociated in the presence of an RGD peptide, as well as of a simple carboxylic acid, suggesting that the RGD aspartyl carboxylate is an essential element that directly interacts with the alpha5 cation-binding domain.

J Biol Chem, 1998 Sep 18, 273(38), 24701 - 7
The insulin-like growth factor (IGF)binding protein 1 binding epitope on IGF-I probed by heteronuclear NMR spectroscopy and mutational analysis; Jansson M et al.; NMR spectroscopy studies and biosensor interaction analysis of native and site-directed mutants of insulin-like growth factor I (IGF-I) was applied to identify the involvement of individual residues in IGF-I binding to IGF-binding protein 1 (IGFBP-1) . Backbone NMR chemical shifts were found to be affected by IGFBP-1 binding in the following residues: Pro2, Glu3, Cys6, Gly7, Gly19, Pro28-Gly30, Gly32, Arg36, Arg37, Gln40-Gly42, Pro63, Lys65, Pro66, and Lys68-Ala70 . Three IGF-I arginine side chains were identified by NMR to participate in IGFBP-1 binding . All IGF-I arginine residues were replaced by alanines, using site-directed mutagenesis, in four single substituted variants, IGF-I(R21A), IGF-I(R50A), IGF-I(R55A), and IGF-I(R56A), and one double replacement mutant, IGF-I(R36A/R37A) . Biosensor interaction analysis binding studies demonstrate the involvement of Arg36-Arg37 and Arg50 in IGFBP-1 binding, while experiments with the IGF-I receptor implicate Arg21, Arg36-Arg37, and Arg56 as part of the receptor binding epitope . These overlapping binding surfaces explain why IGF-I receptor and IGFBP-1 binding to IGF-I is competitive . The C terminus of free, but not IGFBP-1-bound, IGF-I is found to exist in two distinct, NMR-detectable conformations at 30 degreesC . One possible explanation for this structural heterogeneity could be cis-trans isomerization of the Cys6-Cys48 disulfide bond.

J Biol Chem, 1998 Sep 18, 273(38), 24564 - 74
Pre-steady state analysis of the assembly of wild type and mutant circular clamps of Escherichia coli DNA polymerase III onto DNA; Bertram JG et al.; The beta protein, a dimeric ring-shaped clamp essential for processive DNA replication by Escherichia coli DNA polymerase III holoenzyme, is assembled onto DNA by the gamma complex . This study examines the clamp loading pathway in real time, using pre-steady state fluorescent depolarization measurements to investigate the loading reaction and ATP requirements for the assembly of beta onto DNA . Two beta dimer interface mutants, L273A and L108A, and a nonhydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), have been used to show that ATP binding is required for gamma complex and beta to associate with DNA, but that a gamma complex-catalyzed ATP hydrolysis is required for gamma complex to release the beta.DNA complex and complete the reaction . In the presence of ATP and gamma complex, the beta mutants associate with DNA as efficiently as wild type beta . However, completion of the reaction is much slower with the beta mutants because of decreased ATP hydrolysis by the gamma complex, resulting in a much slower release of the mutants onto DNA . The effects of mutations in the dimer interface were similar to the effects of replacing ATP with ATPgammaS in reactions using wild type beta . Thus, the assembly of beta around DNA is coupled tightly to the ATPase activity of the gamma complex, and completion of the assembly process requires ATP hydrolysis for turnover of the catalytic clamp loader.

J Biol Chem, 1998 Sep 18, 273(38), 24550 - 63
ATP binding to the Escherichia coli clamp loader powers opening of the ring-shaped clamp of DNA polymerase III holoenzyme; Hingorani MM et al.; The Escherichia coli gamma complex serves as a clamp loader, catalyzing ATP-dependent assembly of beta protein clamps onto primed DNA templates during DNA replication . These ring-shaped clamps tether DNA polymerase III holoenzyme to the template, facilitating rapid and processive DNA synthesis . This report focuses on the role of ATP binding and hydrolysis catalyzed by the gamma complex during clamp loading . We show that the energy from ATP binding to gamma complex powers several initial events in the clamp loading pathway . The gamma complex (gamma2 delta delta'chi psi) binds two ATP molecules (one per gamma subunit in the complex) with high affinity (Kd = 1-2 . 5 x 10(-6) M) or two adenosine 5'-O-(3-thiotriphosphate)(ATPgammaS) molecules with slightly lower affinity (Kd = 5-6.5 x 10(-6) M) . Experiments performed prior to the first ATP turnover (kcat = 4 x 10(-3) s-1 at 4 degreesC), or in the presence of ATPgammaS (kcat = 1 x 10(-4) s-1 at 37 degreesC), demonstrate that upon interaction with ATP the gamma complex undergoes a change in conformation . This ATP-bound gamma complex binds beta and opens the ring at the dimer interface . Still prior to ATP hydrolysis, the composite of gamma complex and the open beta ring binds with high affinity to primer-template DNA . Thus ATP binding powers all the steps in the clamp loading pathway leading up to the assembly of a gamma complex . open beta ring.DNA intermediate, setting the stage for ring closing and turnover of the clamp loader, steps that may be linked to subsequent hydrolysis of ATP.

J Biol Chem, 1998 Sep 18, 273(38), 24521 - 8
The ortho effect makes manganese(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin a powerful and potentially useful superoxide dismutase mimic; Batinic-Haberle I et al.; The ortho, meta, and para isomers of manganese(III) 5,10,15, 20-tetrakis(N-methylpyridyl)porphyrin, MnTM-2-PyP5+, MnTM-3-PyP5+, and MnTM-4-PyP5+, respectively, were analyzed in terms of their superoxide dismutase (SOD) activity in vitro and in vivo . The impact of their interaction with DNA and RNA on the SOD activity in vivo and in vitro has also been analyzed . Differences in their behavior are due to the combined steric and electrostatic factors . In vitro catalytic activities are closely related to their redox potentials . The half-wave potentials (E1/2) are +0.220 mV, +0.052 mV, and +0.060 V versus normal hydrogen electrode, whereas the rates of dismutation (kcat) are 6.0 x 10(7), 4.1 x 10(6), and 3.8 x 10(6) M-1 s-1 for the ortho, meta, and para isomers, respectively . However, the in vitro activity is not a sufficient predictor of in vivo efficacy . The ortho and meta isomers, although of significantly different in vitro SOD activities, have fairly close in vivo SOD efficacy due to their similarly weak interactions with DNA . In contrast, due to a higher degree of interaction with DNA, the para isomer inhibited growth of SOD-deficient Escherichia coli.

J Biol Chem, 1998 Sep 18, 273(38), 24414 - 9
The Gcn5.Ada complex potentiates the histone acetyltransferase activity of Gcn5; Syntichaki P et al.; The Gcn5 histone acetyltransferase (HAT) is part of a large multimeric complex that is required for transcriptional activation in yeast . This complex can acetylate in vitro and in a Gcn5-dependent manner both nucleosomal and free core histones . For this reason it is believed that part of the function of the Gcn5.Ada complex is chromatin remodeling effected by histone acetylation . The roles of the other subunits of this complex are not yet known . We have generated mutated Gcn5 proteins with severely attenuated in vitro HAT activities . Despite their apparent loss in HAT activity, these GCN5 derivatives complemented all the defects of a gcn5 strain . We have shown that when these mutated proteins were produced in yeast cells in the absence of another component of the complex, Ada2, their activity was still compromised . By contrast, when produced in the wild type context, they were partially capable of acetylating free histones and were even more active when nucleosomal arrays were used as substrates . Kinetic enzymatic analyses showed that the rate of catalysis by Gcn5 was enhanced when the mutated proteins were produced in yeast in the presence of Ada2 . Because Ada2 is required for the assembly of Gcn5, we conclude that one role for components of the Gcn5.Ada complex is the potentiation of its HAT activity.

J Bacteriol, 1998 Sep, 180(18), 4872 - 8
Escherichia coli tol-pal mutants form outer membrane vesicles; Bernadac A et al.; Mutations in the tol-pal genes induce pleiotropic effects such as release of periplasmic proteins into the extracellular medium and hypersensitivity to drugs and detergents . Other outer membrane defective strains such as tolC, lpp, and rfa mutations are also altered in their outer membrane permeability . In this study, electron microscopy and Western blot analyses were used to show that strains with mutations in each of the tol-pal genes formed outer membrane vesicles after growth in standard liquid or solid media . This phenotype was not observed in tolC and rfaD cells in the same conditions . A tolA deletion in three different Escherichia coli strains was shown to lead to elevated amounts of vesicles . These results, together with plasmid complementation experiments, indicated that the formation of vesicles resulted from the defect of any of the Tol-Pal proteins . The vesicles contained outer membrane trimeric porins correctly exposed at the cell surface . Pal outer membrane lipoprotein was also immunodetected in the vesicle fraction of tol strains . The results are discussed in view of the role of the Tol-Pal transenvelope proteins in maintaining outer membrane integrity by contributing to target or integrate newly synthesized components of this structure.

J Bacteriol, 1998 Sep, 180(18), 4865 - 71
Mutational analysis of the transcriptional regulator GcvA: amino acids important for activation, repression, and DNA binding; Jourdan AD et al.; The GcvA protein is required for both glycine-mediated activation and purine-mediated repression of the gcvTHP operon . Random and site-directed PCR mutagenesis was used to create nucleotide changes in gcvA to identify residues of the protein involved in activation, repression, and DNA binding . Single amino acid substitutions at L30 and F31 cause a defect in activation of a gcvT-lacZ fusion but have no effect on repression or DNA binding . Single amino acid substitutions at V32 and S38 cause the loss of binding of GcvA to DNA . A deletion of the carboxy-terminal 14 amino acids of GcvA results in the loss of purine-mediated repression and, consequently, a constitutive activation of a gcvT-lacZ fusion . The results of this study partially define regions of GcvA involved in activation, repression, and DNA binding and demonstrate that these functions of GcvA are genetically separable.

J Bacteriol, 1998 Sep, 180(18), 4843 - 9
The ggpS gene from Synechocystis sp . strain PCC 6803 encoding glucosyl-glycerol-phosphate synthase is involved in osmolyte synthesis; Marin K et al.; A salt-sensitive mutant of Synechocystis sp . strain PCC 6803 defective in the synthesis of the compatible solute glucosylglycerol (GG) was used to search for the gene encoding GG-phosphate synthase (GGPS), the key enzyme in GG synthesis . Cloning and sequencing of the mutated region and the corresponding wild-type region revealed that a deletion of about 13 kb occurred in the genome of mutant 11 . This deletion affected at least 10 open reading frames, among them regions coding for proteins showing similarities to trehalose (otsA homolog)- and glycerol-3-phosphate-synthesizing enzymes . After construction and characterization of mutants defective in these genes, it became obvious that an otsA homolog (sll1566) (T . Kaneko et al., DNA Res . 3:109-136, 1996) encodes GGPS, since only the mutant affected in sll1566 showed salt sensitivity combined with a complete absence of GG accumulation . Furthermore, the overexpression of sll1566 in Escherichia coli led to the appearance of GGPS activity in the heterologous host . The overexpressed protein did not show the salt dependence that is characteristic for the GGPS in crude protein extracts of Synechocystis.

J Bacteriol, 1998 Sep, 180(18), 4828 - 33
Phosphorylation of the periplasmic binding protein in two transport systems for arginine incorporation in Escherichia coli K-12 is unrelated to the function of the transport system; Celis RT et al.; In Escherichia coli K-12, the accumulation of arginine is mediated by two distinct periplasmic binding protein-dependent transport systems, one common to arginine and ornithine (AO system) and one for lysine, arginine, and ornithine (LAO system) . Each of these systems includes a specific periplasmic binding protein, the AO-binding protein for the AO system and the LAO-binding protein for the LAO system . The two systems include a common inner membrane transport protein which is able to hydrolyze ATP and also phosphorylate the two periplasmic binding proteins . Previously, a mutant resistant to the toxic effects of canavanine, with low levels of transport activities and reduced levels of phosphorylation of the two periplasmic binding proteins, was isolated and characterized (R . T . F . Celis, J . Biol . Chem . 265:1787-1793, 1990) . The gene encoding the transport ATPase enzyme (argK) has been cloned and sequenced . The gene possesses an open reading frame with the capacity to encode 268 amino acids (mass of 29.370 Da) . The amino acid sequence of the protein includes two short sequence motifs which constitute a well-defined nucleotide-binding fold (Walker sequences A and B) present in the ATP-binding subunits of many transporters . We report here the isolation of canavanine-sensitive derivatives of the previously characterized mutant . We describe the properties of these suppressor mutations in which the transport of arginine, ornithine, and lysine has been restored . In these mutants, the phosphorylation of the AO- and LAO-binding proteins remains at a low level . This information indicates that whereas hydrolysis of ATP by the transport ATPase is an obligatory requirement for the accumulation of these amino acids in E . coli K-12, the phosphorylation of the periplasmic binding protein is not related to the function of the transport system.

J Bacteriol, 1998 Sep, 180(18), 4799 - 803
Probing the role of cysteine residues in glucosamine-1-phosphate acetyltransferase activity of the bifunctional GlmU protein from Escherichia coli: site-directed mutagenesis and characterization of the mutant enzymes; Pompeo F et al.; The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme from Escherichia coli was lost when GlmU was stored in the absence of beta-mercaptoethanol or incubated with thiol-specific reagents . The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain . To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins . Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively . The Km values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding . The uridyltransferase activities of wild-type and mutant GlmU proteins were similar . From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.

J Bacteriol, 1998 Sep, 180(18), 4790 - 8
Mutational inactivation of a gene homologous to Escherichia coli ptsP affects poly-beta-hydroxybutyrate accumulation and nitrogen fixation in Azotobacter vinelandii; Segura D et al.; Strain DS988, an Azotobacter vinelandii mutant with a reduced capacity to accumulate poly-beta-hydroxybutyrate, was isolated after mini-Tn5 mutagenesis of the UW136 strain . Cloning and nucleotide sequencing of the affected locus revealed a gene homologous to Escherichia coli ptsP which encodes enzyme INtr, a homologue of enzyme I of the phosphoenol pyruvate-sugar phosphotransferase system with an N-terminal domain similar to the N-terminal domain of some NifA proteins . Strain DS988 was unable to grow diazotrophically with 10 mM glucose as a carbon source . Diazotrophic growth on alternative carbon sources such as gluconate was only slightly affected . Glucose uptake, as well as glucose kinase and glucose-6-phosphate-dehydrogenase activities that lead to the synthesis of gluconate-6-phosphate, were not affected by the ptsP mutation . The inability of DS988 to grow diazotrophically in 10 mM glucose was overcome by supplying ammonium or other sources of fixed nitrogen . Acetylene reduction activity but not transcription of the nitrogenase structural gene nifH was shown to be impaired in strain DS988 when it was incubated in 10 mM glucose . The diazotrophic growth defect of DS988 was restored either by increasing the glucose concentration to above 20 mM or by lowering the oxygen concentration . These data suggest that a mutation in ptsP leads to a failure in poly-beta-hydroxybutyrate metabolism and in the respiratory protection of nitrogenase under carbon-limiting conditions.

J Bacteriol, 1998 Sep, 180(18), 4781 - 9
Characterization of native and recombinant forms of an unusual cobalt-dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeon Pyrococcus furiosus; Ghosh M et al.; Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography . The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit . Its catalytic activity also required the addition of Co2+ ions (Kd, 0.24 mM), indicating that the enzyme has a second metal ion binding site . Co2+ could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+ . The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus . Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100 degrees C . The N-terminal amino acid sequence of the purified prolidase was used to identify in the P . furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids . This gene was expressed in Escherichia coli and the recombinant protein was purified . Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P . furiosus . Furthermore, the Km values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater Vmax value than the native protein . The amino acid sequence of P . furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors . The P . furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.

J Mol Biol, 1998 Sep 11, 282(1), 149 - 65
GlnK, a PII-homologue: structure reveals ATP binding site and indicates how the T-loops may be involved in molecular recognition; Xu Y et al.; GlnK is a recently discovered homologue of the PII signal protein, an indicator of the nitrogen status of bacteria . PII occupies a central position in the dual cascade that regulates the activity of glutamine synthetase and the transcription of its gene . The complete role of Escherichia coli GlnK is yet to be determined, but already it is known that GlnK behaves like PII and can substitute for PII under some circumstances thereby adding to the subtleties of nitrogen regulation . There are also indications that the roles of the two proteins differ; the expression of PII is constitutive while that of GlnK is linked to the level of nitrogen in the cell . The discovery of GlnK begs the question of why E . coli has both GlnK and PII . Clearly, the structural similarities and differences of GlnK and PII will lead to a better understanding of how PII-like proteins function in E . coli and other organisms.We have crystallised and solved the X-ray structure of GlnK at 2.0 A resolution . The asymmetric unit has two independent copies of the GlnK subunit and both pack around 3-fold axes to form trimers . The trimers have a barrel-like core with recognition loops (the T-loops) that protrude from the top of the molecule . The two GlnK molecules have similar core structures to PII but differ significantly at the C terminus and the loops . The T-loops of the two GlnK molecules also differ from each other; one is disordered while the conformation of the other is stabilised by lattice contacts . The conformation of the ordered T-loop of GlnK differs from that observed in the PII structure despite the fact that their sequences are very similar . The structures suggest that the T-loops do not have a rigid structure and that they may be flexible in solution . The presence of a turn of 310 helix in the middle of the T-loop suggests that secondary structure could form when it interacts with soluble receptor enzymes.Co-crystals of GlnK and ATP were used to determine the structure of the complex . In these crystals, GlnK occupies a position of 3-fold symmetry . ATP binds in a cleft on the side of the molecule . The cleft is suitably positioned for ATP to influence the flexible T-loops . It is found at the junction of two beta sheets and is formed by two peptides one of which contains a variant of the "Gly-loop" found in other mononucleotide binding proteins . This sequence, Thr-Gly-X-X-Gly-Asp-Gly-Lys-Ile-Phe, forms part of the B-loop and is conserved in a wide variety of organisms that include bacteria, algae and archeabacteria . This sequence is more highly conserved than the functional T-loop, suggesting that ATP has an important role in PII-like proteins .

J Mol Biol, 1998 Sep 11, 282(1), 25 - 41
Roles of pIII in filamentous phage assembly; Rakonjac J et al.; Filamentous phage protein III (pIII), located at one end of the phage, is required for infectivity and stability of the particle . Cells infected with phage from which gene III has been completely deleted produce particles that are not released into the medium but stay associated at the surface . These particles are much longer than normal phage . They can be released by subsequent expression of pIII . Viewed with the electron microscope, cells infected with gene III deletion phage are decorated with structures that resemble extremely long pili . Surprisingly, such cells are viable and can form colonies . The pIII deficiency can be complemented in trans, but there is a threshold concentration below which assembly does not occur . Above this threshold, pIII is used very efficiently and is incorporated into infectious but longer than unit length phage . As the concentration of pIII is increased, the number of infectious particles increases, and their average length decreases.pIII stabilizes pVI, a second phage protein found at the pIII end of the particle . In the absence of pIII, degradation of pVI is very rapid . pIII is thus not only required for infectivity and particle stability, but to terminate assembly and release the phage from its assembly site .

Plant Physiol, 1998 Sep, 118(1), 275 - 83
Isolation and characterization of a histidine biosynthetic gene in Arabidopsis encoding a polypeptide with two separate domains for phosphoribosyl-ATP pyrophosphohydrolase and phosphoribosyl-AMP cyclohydrolase; Fujimori K et al.; Phosphoribosyl-ATP pyrophosphohydrolase (PRA-PH) and phosphoribosyl-AMP cyclohydrolase (PRA-CH) are encoded by HIS4 in yeast and by hisIE in bacteria and catalyze the second and the third step, respectively, in the histidine biosynthetic pathway . By complementing a hisI mutation of Escherichia coli with an Arabidopsis cDNA library, we isolated an Arabidopsis cDNA (At-IE) that possesses these two enzyme activities . The At-IE cDNA encodes a bifunctional protein of 281 amino acids with a calculated molecular mass of 31,666 D . Genomic DNA-blot analysis with the At-IE cDNA as a probe revealed a single-copy gene in Arabidopsis, and RNA-blot analysis showed that the At-IE gene was expressed ubiquitously throughout development . Sequence comparison suggested that the At-IE protein has an N-terminal extension of about 50 amino acids with the properties of a chloroplast transit peptide . We demonstrated through heterologous expression studies in E . coli that the functional domains for the PRA-CH (hisI) and PRA-PH (hisE) resided in the N-terminal and the C-terminal halves, respectively, of the At-IE protein.

Science, 1998 Sep 11, 281(5383), 1666 - 8
Grain feeding and the dissemination of acid-resistant Escherichia coli from cattle; Diez-Gonzalez F et al.; The gastric stomach of humans is a barrier to food-borne pathogens, but Escherichia coli can survive at pH 2.0 if it is grown under mildly acidic conditions . Cattle are a natural reservoir for pathogenic E . coli, and cattle fed mostly grain had lower colonic pH and more acid-resistant E . coli than cattle fed only hay . On the basis of numbers and survival after acid shock, cattle that were fed grain had 10(6)-fold more acid-resistant E . coli than cattle fed hay, but a brief period of hay feeding decreased the acid-resistant count substantially.

J Med Chem, 1998 Sep 10, 41(19), 3572 - 81
Prodrugs of anthracyclines for use in antibody-directed enzyme prodrug therapy; Florent JC et al.; A series of new prodrugs of daunorubicin and doxorubicin which are candidates for antibody-directed enzyme prodrug therapy (ADEPT) is reported . These compounds (25a,b,c and 32a,b,c) have been designed to generate cytotoxic drugs after activation with beta-glucuronidase . As expected, recovery of the active drug was observed after enzymatic cleavage by Escherichia coli beta-glucuronidase as well as by a fusion protein which has been obtained from human beta-glucuronidase and humanized CEA-specific binding region . The six prodrugs are highly stable and are more than 100-fold less cytotoxic than doxorubicin against murine L1210 cell lines . The ortho-substituted phenyl carbamates 25a,b,c are better substrates for beta-glucuronidase than the corresponding para-substituted analogues . After taking into account additional factors such as stability in plasma and kinetics of enzymatic cleavage, we selected the o-nitro prodrug 25c for clinical trials.

J Androl, 1998 Jul-Aug, 19(4), 385 - 93
SP22: a novel fertility protein from a highly conserved gene family; Welch JE et al.; Three nucleotide sequences encoding SP22, a protein originally identified in detergent extracts of cauda epididymal sperm, were isolated from a rat testis cDNA library . While two of these cDNA sequences differed only in their 5' untranslated regions, a third cDNA was predicted to contain an additional 13 amino acids of coding sequence . Amino acid sequences obtained following Edman degradation of purified SP22 protein and cDNA sequence data both indicated that SP22 was a member of a highly conserved and widely expressed gene family found in organisms as diverse as human and Escherichia coli . Interestingly, while a 1-kb mRNA transcript was widely expressed in somatic tissues, a unique pattern of testicular expression was observed, including the appearance of a novel 1.5-kb transcript and an increase in the abundance of the 1-kb transcript during spermatogenic cell development . Anti-SP22 peptide antiserum was shown to recognize a family of 22-kDa proteins on western blots of detergent-extracted cauda epididymal sperm protein, suggesting that multiple charge variants of SP22 coexist . Moreover, affinity-purified anti-SP22 peptide immunoglobulin localized in a highly specific manner to the anterior-ventral surface of the equatorial segment of the sperm head . This is an extremely intriguing finding as SP22 was originally shown to be highly correlated with, and predictive of, the fertilizing ability of cauda epididymal sperm . Although no conclusive function has been attributed to any members of the SP22 gene family, the localization of SP22 over a discrete region of the sperm head suggests a pivotal role in sperm-egg interactions.

Curr Microbiol, 1998 Oct, 37(4), 226 - 30
Changes in protein synthesis as a consequence of heme depletion in Escherichia coli; Rompf A et al.; Two-dimensional gel electrophoresis and N-terminal amino acid sequence determination were used to compare the protein synthesis of exponentially growing Escherichia coli with heme-deficient cells . Mutation of the E . coli hemA gene encoding glutamyl-tRNA reductase resulted in the absence of detectable amounts of heme . As a consequence of heme deficiency, the induction of tryptophanase (trpA), citrate synthase (gltA), and aldehyde dehydrogenase (aldA) and the repression of enolase (eno) and phosphoglycerate kinase (pgk) were observed . All induced genes are under the control of the catabolite repressor protein Crp . The observed changes in gene expression as a consequence of heme depletion are discussed.

J Mol Evol, 1998 Sep, 47(3), 363 - 8
Heterologous gene expression in an Escherichia coli population under starvation stress conditions; Fani R et al.; A novel system to study the evolution of transcription signals in heterologous systems under selective starvation conditions is described . It is based on the plasmid-mediated transfer of his biosynthetic genes from Azospirillum brasilense into a heterologous Escherichia coli mutant population lacking histidine biosynthetic ability . We show that under highly selective stressful conditions, genetic changes in the donor plasmid lead to mutated sequences that are efficiently recognized as promoters by the E . coli RNA polymerase.

J Mol Evol, 1998 Sep, 47(3), 343 - 52
Identification and expression of the SOS response, aidB-like, gene in the marine sponge Geodia cydonium: implication for the phylogenetic relationships of metazoan acyl-CoA dehydrogenases and acyl-CoA oxidases; Krasko A et al.; Sponges (Porifera) are the phylogenetically oldest metazoan organisms . From one member of the siliceous sponges, Geodia cydonium, the cDNA encoding a putative SOS protein, the AidB-like protein of the Ada system from bacteria, was isolated and characterized . The cDNA, GCaidB, comprises an open reading frame of 446 amino acid (aa) residues encoding a polypeptide with a calculated Mr of 49,335 . This molecule shows high similarity to the bacterial AidB proteins from Mycobacterium tuberculosis and Escherichia coli and somewhat lower similarities to acyl-CoA dehydrogenases (ADHs) and acyl-CoA oxidases (AOXs) . Northern blot analysis confirmed the presence of the complete transcript . The deduced sponge aa sequence, GC_aidB, possesses the two characteristic acyl-CoA dehydrogenase signatures 1 and 2 . Incubation of the sponge with N-methyl-N'-nitro-N-nitrosoguanidine causes a strong increase in the 2.1-kb large transcript of GCaidB; maximal expression is seen after 24 h of incubation with this DNA methylating agent . ADHs and AOXs can be grouped, depending on the position of the catalytically important Glu residue, into the Glu-Gly (Glu adjacent to Gly) class and the Glu-Arg (Glu adjacent to Arg) class . The phylogenetically oldest metazoan AidB-like molecule, GC_aidB of G . cydonium, belongs to the Glu-Gly class of ADHs . Phylogenetic analyses of the Glu-Gly class enzymes, with the described AidB-like protein from G . cydonium and the bacterial AidB polypeptides, together with metazoan ADHs and AOXs, revealed that the AidB(-like) proteins diverged first from a common ancestor, while the eukaryotic AOX and ADA polypeptides as well as the GHDs appeared later . According to the analyses, the very long-chain ADHs are older than the medium-chain, short-chain, and branched-chain ADHs . Inclusion of the phylogenetical oldest member of the Glu-Arg class of enzymes, the bacterial ADH-CaiA sequence in these analyses, revealed that this class of enzymes appeared later in evolution and arose from the Glu-Gly class perhaps after gene duplication.

J Mol Evol, 1998 Sep, 47(3), 268 - 74
An evaluation of measures of synonymous codon usage bias; Comeron JM et al.; Synonymous codons are not generally used at equal frequencies, and this trend is observed for most genes and organisms . Several methods have been proposed and used to estimate the degree of the nonrandom use of the different synonymous codons . The estimates obtained by these methods, however, show different levels of both precision and dispersion when coding regions of a finite number of codons are under analysis . Here, we present a study, based on computer simulation, of how the different methods proposed to evaluate the nonrandom use of synonymous codons are affected by the length of the coding region analyzed . The results show that some of these methods are heavily influenced by the number of codons and that the comparison of codon usage bias between coding regions of different lengths shows a methodological bias under different conditions of nonrandom use of synonymous codons . The study of the dispersion of the estimates obtained by the different methods gives, on the other hand, an indication of the methods to be applied to compare values of codon usage bias among coding regions of equivalent length.

Arch Microbiol, 1998 Oct, 170(4), 259 - 68
Mutations in cell division proteins FtsZ and FtsA inhibit phiX174 protein-E-mediated lysis of Escherichia coli; Witte A et al.; Electron microscopic studies emphasized that the protein-E-specific transmembrane tunnel structure, which permeabilizes Escherichia coli, is not randomly distributed over the cell envelope but is restricted to areas of potential division sites . These sites were located predominantly in the middle of the cell, but approximately one-third of these structures are found at the polar sites . Therefore, E . coli mutant strains with defects in cell division components were tested for their sensitivity to protein-E-mediated lysis . The ftsZ84 and the ftsA12 cell division mutant strains of E . coli were tolerant to protein-E-mediated lysis, whereas the ftsA3 mutant strain was lysed by protein E under conditions nonpermissive for division . The protein-E-tolerant phenotype of ftsZ84 and ftsA12 and the lysis-sensitive phenotype of other components of the septosome (e.g., ftsA3, ftsQ, and ftsI) suggest that initiation of cell division - rather than specific functions of cell division - plays an essential role in protein-E-mediated lysis . SulA-overproducing cells had a lysis-positive phenotype, the ring structure - but not the GTPase function - of FtsZ was impaired.

Nat Genet, 1998 Sep, 20(1), 51 - 3
Mutations in a polycistronic nuclear gene associated with molybdenum cofactor deficiency; Reiss J et al.; All molybdoenzymes other than nitrogenase require molybdopterin as a metal-binding cofactor . Several genes necessary for the synthesis of the molybdenum cofactor (MoCo) have been characterized in bacteria and plants . The proteins encoded by the Escherichia coli genes moaA and moaC catalyse the first steps in MoCo synthesis . The human homologues of these genes are therefore candidate genes for molybdenum cofactor deficiency, a rare and fatal disease . Using oligonucleotides complementary to a conserved region in the moaA gene, we have isolated a human cDNA derived from liver mRNA . This transcript contains an open reading frame (ORF) encoding the human moaA homologue and a second ORF encoding a human moaC homologue . Mutations can be found in the majority of MoCo-deficient patients that confirm the functional role of both ORFs in the corresponding gene MOCS1 (for 'molybdenum cofactor synthesis-step 1') . Northern-blot analysis detected only full-length transcripts containing both consecutive ORFs in various human tissues . The mRNA structure suggests a translation reinitiation mechanism for the second ORF . These data indicate the existence of a eukaryotic mRNA, which as a single and uniform transcript guides the synthesis of two different enzymatic polypeptides with disease-causing potential.

Ophthalmic Res, 1998, 30(6), 388 - 93
Uveal mast cells are not required for rodent uveitis; Smith JR et al.; Uveal mast cells have previously been considered to be vital mediators of experimental uveitis . We extended the study of these cells to experimental melanin-induced uveitis (EMIU), a recently described clinically relevant model, and re-examined their role in endotoxin-induced uveitis (EIU) using genetically mast cell-depleted mice on a single background . EMIU was induced in Fischer 344 rats by immunisation with bovine ocular melanin (250 microgram . Animals were killed immediately, and on days 1 and 3 of clinical disease . Numbers of uveal mast cells and the percentage of degranulated cells were counted in whole-mount preparations . There was no significant change in either measure across the selected time points . To induce EIU, normal and mast cell-depleted DBA/2 mice were injected with Escherichia coli lipopolysaccharide (400 microgram) . Cells infiltrating the eye 24 h after injection were quantified in 5 micrometer ocular cross-sections . Disease was not significantly reduced in the mast cell-depleted mutants . We conclude that uveal mast cells are not required for the development of EMIU or, in contrast to earlier work, EIU.

J Cell Biol, 1998 Sep 7, 142(5), 1381 - 91
Endothelial cell E- and P-selectin and vascular cell adhesion molecule-1 function as signaling receptors; Lorenzon P et al.; Previous studies have shown that polymorphonuclear leukocyte (PMN) adherence to endothelial cells (EC) induces transient increases in EC cytosolic free calcium concentration ({Ca2+}i) that are required for PMN transit across the EC barrier (Huang, A.J., J.E . Manning, T . M . Bandak, M.C . Ratau, K.R . Hanser, and S.C . Silverstein . 1993 . J . Cell Biol . 120:1371-1380) . To determine whether stimulation of {Ca2+}i changes in EC by leukocytes was induced by the same molecules that mediate leukocyte adherence to EC, {Ca2+}i was measured in Fura2-loaded human EC monolayers . Expression of adhesion molecules by EC was induced by a pretreatment of the cells with histamine or with Escherichia coli lipopolysaccharide (LPS), and {Ca2+}i was measured in single EC after the addition of mAbs directed against the EC adhesion proteins P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or platelet/endothelial cell adhesion molecule-1 (PECAM-1) . Both anti-P- and anti-E-selectin mAb, as well as anti-VCAM-1 mAb, induced transient increases in EC {Ca2+}i that were comparable to those induced by 200 microM histamine . In contrast, no effect was obtained by mAbs directed against the endothelial ICAM-1 or PECAM-1 . PMN adherence directly stimulated increases in {Ca2+}i in histamine- or LPS-treated EC . mAbs directed against leukocyte CD18 or PECAM-1, the leukocyte counter-receptors for endothelial ICAM-1 and PECAM-1, respectively, did not inhibit PMN-induced EC activation . In contrast, mAb directed against sialyl Lewis x (sLex), a PMN ligand for endothelial P- and E-selectin, completely inhibited EC stimulation by adherent PMN . Changes in EC {Ca2+}i were also observed after adherence of peripheral blood monocytes to EC treated with LPS for 5 or 24 h . In these experiments, the combined addition of mAbs to sLex and VLA-4, the leukocyte counter-receptor for endothelial VCAM-1, inhibited {Ca2+}i changes in the 5 h-treated EC, whereas the anti-VLA-4 mAb alone was sufficient to inhibit {Ca2+}i changes in the 24 h-treated EC . Again, no inhibitory effect was observed with an anti-CD18 or anti-PECAM-1 mAb . Of note, the conditions that induced changes in EC {Ca2+}i, i.e . , mAbs directed against endothelial selectins or VCAM-1, and PMN or monocyte adhesion to EC via selectins or VCAM-1, but not via ICAM-1 or PECAM-1, also induced a rearrangement of EC cytoskeletal microfilaments from a circumferential ring to stress fibers . We conclude that, in addition to their role as adhesion receptors, endothelial selectins and VCAM-1 mediate endothelial stimulation by adhering leukocytes.

Genes Dev, 1998 Sep 1, 12(17), 2782 - 90
Sequence and DNA structural determinants of N4 virion RNA polymerase-promoter recognition; Dai X et al.; Coliphage N4-coded, virion-encapsidated RNA polymerase (vRNAP) is able to bind to and transcribe promoter-containing double-stranded DNAs when the template is supercoiled and Escherichia coli single-stranded DNA-binding protein (Eco SSB) is present . We report that vRNAP-promoter recognition and activity on these templates require specific sequences and a hairpin structure on the template strand . Hairpin extrusion, induced by Mg(II) and physiological superhelical density, is essential to provide the correct DNA structure for polymerase recognition, as mutant promoters that do not form hairpins show reduced in vitro activity . Therefore, a supercoil-induced DNA structural transition regulates N4 vRNAP transcription . Eco SSB activates transcription at physiological superhelical densities by stabilizing the template-strand hairpin . Specific sequences at the promoters are conserved to provide proper contacts for vRNAP, to support hairpin extrusion, or both . We propose a model for in vivo utilization of the vRNAP promoters, and discuss the roles of DNA supercoiling and Eco SSB in promoter activation.

Genes Dev, 1998 Sep 1, 12(17), 2770 - 81
Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome; Vanzo NF et al.; The Escherichia coli RNA degradosome is the prototype of a recently discovered family of multiprotein machines involved in the processing and degradation of RNA . The interactions between the various protein components of the RNA degradosome were investigated by Far Western blotting, the yeast two-hybrid assay, and coimmunopurification experiments . Our results demonstrate that the carboxy-terminal half (CTH) of ribonuclease E (RNase E) contains the binding sites for the three other major degradosomal components, the DEAD-box RNA helicase RhlB, enolase, and polynucleotide phosphorylase (PNPase) . The CTH of RNase E acts as the scaffold of the complex upon which the other degradosomal components are assembled . Regions for oligomerization were detected in the amino-terminal and central regions of RNase E . Furthermore, polypeptides derived from the highly charged region of RNase E, containing the RhlB binding site, stimulate RhlB activity at least 15-fold, saturating at one polypeptide per RhlB molecule . A model for the regulation of the RhlB RNA helicase activity is presented . The description of RNase E now emerging is that of a remarkably complex multidomain protein containing an amino-terminal catalytic domain, a central RNA-binding domain, and carboxy-terminal binding sites for the other major components of the RNA degradosome.

Genes Dev, 1998 Sep 1, 12(17), 2673 - 83
Phosphorylation regulates association of the transcription factor Pho4 with its import receptor Pse1/Kap121; Kaffman A et al.; The transcription factor Pho4 is phosphorylated and localized predominantly to the cytoplasm when budding yeast are grown in phosphate-rich medium and is unphosphorylated and localized to the nucleus upon phosphate starvation . We have investigated the requirements for nuclear import of Pho4 and find that Pho4 enters the nucleus via a nonclassical import pathway that utilizes the importin beta family member Pse1/Kap121 . Pse1 binds directly to Pho4 and is required for its import in vivo . We have defined the nuclear localization signal on Pho4 and demonstrate that it is required for Pse1 binding in vitro and is sufficient for PSE1-dependent import in vivo . Phosphorylation of Pho4 inhibits its interaction with Pse1, providing a mechanism by which phosphorylation may regulate import of Pho4 in vivo.

Nitric Oxide, 1998, 2(3), 203 - 12
Human and rat neutrophils constitutively express neural nitric oxide synthase mRNA; Greenberg SS et al.; Freshly isolated rat circulating neutrophils (PMN) constitutively expressed neural nitric oxide synthase (nNOS) mRNA and nNOS protein and exhibited spontaneous basal release of low concentrations of nitrate and nitrite anion (RNI) . In contrast, rat peripheral monocytes and macrophages were devoid of nNOS mRNA and protein and did not exhibit basal or spontaneous release of RNI . Constitutive neural NOS mRNA was also found in human PMN . However, nNOS protein was not expressed and spontaneous generation of RNI was absent in the human PMN . Spontaneous release of RNI from rat PMN was inhibited by 7-nitroindazole but not by L-N-iminoethyllysine, which further supported the nNOS origin of the spontaneously produced RNI . Intravenous administration of Escherichia coli endotoxin (0.6 mg/kg) did not acutely affect the content of nNOS mRNA or protein but inhibited nNOS-derived production of RNI in PMN and up-regulated iNOS mRNA and iNOS protein in PMN, macrophages, and monocytes . This communication demonstrates the existence of nNOS mRNA in rat and human PMN and nNOS protein in rat PMN . Moreover, the data also show that the nNOS system in rat PMN is functional and is inhibitable by the nNOS inhibitor 7-nitroindazole . These findings offer an explanation for the spontaneous formation of the PMN-derived relaxing factor resembling nitric oxide (NO) . Moreover, since basal production of NO can affect expression of adhesion molecules and cell-cell binding, the nNOS system within the rat may play an important role in PMN function in normal and disease states . Finally and speculatively, if constitutively expressed nNOS mRNA is subject to activation and translation into nNOS protein, nNOS may also play a role in the function of human PMN.

Stroke, 1998 Sep, 29(9), 1959 - 65; discussion 1965-6
Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery after experimental subarachnoid hemorrhage; Onoue H et al.; BACKGROUND AND PURPOSE: Gene transfer with recombinant viral vectors encoding vasodilator proteins may be useful in therapy of cerebral vasospasm after subarachnoid hemorrhage (SAH) . Relaxations mediated by nitric oxide are impaired in cerebral arteries affected by SAH . The present study was designed to determine the effect of SAH on the efficiency of ex vivo adenovirus-mediated gene transfer to canine basilar arteries and to examine whether expression of recombinant endothelial nitric oxide synthase (eNOS) gene may have functional effects on vasomotor reactivity of spastic arteries affected by SAH . METHODS: Replication-deficient recombinant adenovirus vectors encoding bovine eNOS (AdCMVeNOS) and Escherichia coli beta-galactosidase (AdCMVbeta-Gal) genes were used for ex vivo gene transfer . Rings of basilar arteries obtained from control dogs and dogs exposed to SAH were incubated with the vectors in minimum essential medium . Twenty-four hours after gene transfer, expression and function of the recombinant genes were evaluated by (1) histochemical or immunohistochemical staining, (2) beta-galactosidase protein measurement, and (3) isometric tension recording . RESULTS: Transduction with AdCMVbeta-Gal and AdCMVeNOS resulted in the expression of recombinant beta-galactosidase and eNOS proteins mostly in the vascular adventitia . The expression of beta-galactosidase protein was approximately 2-fold higher in SAH arteries than in normal arteries . Endothelium-dependent relaxations caused by bradykinin and substance P were suppressed in SAH arteries . The relaxations to bradykinin were significantly augmented in both normal and SAH arteries after AdCMVeNOS transduction but not after AdCMVbeta-Gal transduction . The relaxations to substance P were augmented by AdCMVeNOS transduction only in normal arteries . Bradykinin and substance P caused relaxations even in endothelium-denuded arteries, when the vessels were transduced with AdCMVeNOS . These endothelium-independent (adventitia-dependent) relaxations to bradykinin observed after AdCMVeNOS transduction were similar between normal and SAH arteries, whereas those to substance P were significantly reduced in SAH arteries compared with normal arteries . CONCLUSIONS: These results suggest that expression of recombinant proteins after adenovirus-mediated gene transfer may be enhanced in cerebral arteries affected by SAH and that successful eNOS gene transfer to spastic arteries can at least partly restore the impaired nitric oxide-mediated relaxations through local (adventitial) production of nitric oxide.

Biochem Biophys Res Commun, 1998 Aug 28, 249(3), 933 - 7
Structural and functional consequences of substitutions at the Pro108-Arg14 hydrogen bond in bovine adrenodoxin; Grinberg A et al.; Elimination of Pro108 in bovine adrenodoxin is known to result in the formation of a misfolded protein that is not able to incorporate a {2Fe-2S} cluster and rapidly degrades upon expression in E . coli . However, no experimental explanation for this phenomenon has been demonstrated so far . Using the recently obtained 3D structure of the truncated mutant Adx(4-108) we have studied the reasons of the protein stabilization by the proline residue by means of site-directed muta-genesis . Two main results have been obtained (i) the conserved hydrogen bond Pro108-Arg14, that connects different structural domains of Adx, contributes 6 kJ/mol into the protein stability and (ii) the presence of proline at position 108 provides a low conformational entropy of the unfolded state, supporting a gain in the Gibbs energy of 5.4 kJ/mol at 37 degrees C.

Biochem Biophys Res Commun, 1998 Aug 28, 249(3), 791 - 6
Identification of stathmin as a novel substrate for p38 delta; Parker CG et al.; p38 mitogen-activated protein kinases (MAPK) are a family of kinases that are activated by cellular stresses and inflammatory cytokines . Although there are many similarities shared by the isoforms of p38 (alpha, beta, gamma, and delta), p38 delta differs from the others in some respects such as inhibitor sensitivity and substrate specificity . Utilizing in a solution kinase assay, we identified a novel p38 delta substrate as stathmin . Stathmin is a cytoplasmic protein that was previously reported to be a substrate of several intracellular signaling kinases and has recently been linked to regulation of microtubule dynamics . p38 delta has significantly higher in vitro phosphorylating activity against stathmin than other p38 isoforms or related MAPKs . In transient expression studies, we found that in addition to different stimuli osmotic stress activates p38 delta to phosphorylate stathmin . The sites of phosphorylation were mapped to Ser-25 and Ser-38, both in vitro and in cells.

Biochem Biophys Res Commun, 1998 Aug 28, 249(3), 773 - 80
Exploration of the structural environment of the iron-sulfur cluster in putidaredoxin by nitrogen-15 NMR spectroscopy of selectively labeled cysteine residues; Sari N et al.; Putidaredoxin is a di-iron protein whose paramagnetic region is not well characterized by 1H detected NMR . We have studied the structure of this region in greater detail by directly observed 15N NMR of oxidized and reduced putidaredoxin preparations in which the six cysteine residues are selectively labeled with 15N . A new method for preparation of a stable form of reduced putidaredoxin has been developed for use in NMR . The 15N NMR spectra of the oxidized and reduced forms are characteristically different, and we have measured and compared 15N chemical shifts, spin-lattice relaxation times (T1), and chemical shift/temperature dependences for both forms . Evidence for localized valencies of the iron atoms in the reduced form is presented . From the 15N T1 values of the oxidized form, reduced distances of the cysteine backbone 15N nuclei from the center of the Fe2S2 cluster have been calculated . These distances are consistent with those calculated from X-ray crystal structure data for five ferredoxins, and confirm the structural similarity of the Fe2S2 clusters in putidaredoxin and in these ferredoxins in the oxidized state.

Am J Respir Crit Care Med, 1998 Sep, 158(3), 840 - 5
Aerosolized surfactant improves pulmonary function in endotoxin-induced lung injury; Lutz C et al.; Surfactant dysfunction is a primary pathophysiologic component in patients with adult respiratory distress syndrome (ARDS) . In this study we tested the efficacy of aerosolized surfactant (Sf ) replacement in a severe lung injury model of endotoxin-induced ARDS . Twenty-one certified healthy pigs were anesthetized, surgically prepared for measurement of hemodynamic and lung function, then randomized into one of four groups: (1) control (n = 5), surgical instrumentation only; (2) lipopolysaccharide (LPS) (n = 6), infused with Escherichia coli LPS (100 microgram/kg) without positive end- expiratory pressure (PEEP) and ventilated with a nonhumidified gas mixture of 50% N2O and 50% O2; (3) LPS + PEEP (n = 4), infused with LPS, placed on PEEP (7.5 cm H2O), and ventilated with a humidified gas mixture; and (4) LPS + PEEP + Sf (n = 6), infused with LPS, placed on PEEP, and ventilated with aerosolized Sf (Infasurf, ONY, Inc.) . All animals were studied for 6 h . Arterial PO2 significantly decreased in both the LPS and LPS + PEEP groups (LPS + PEEP = 74 +/- 19 mm Hg; LPS = 74 +/- 19 mm Hg, p < 0.05) while venous admixture (Q S/Q T) increased in these groups (LPS + PEEP = 43.3 +/- 3.9%; LPS = 47.7 +/- 11%, p < 0.05) as compared with the control group . PEEP + Sf reduced the fall in PO2 (142 +/- 20 mm Hg) and rise in Q S/Q T (15.1 +/- 3.6%) caused by LPS . Delayed induction of PEEP (2 h following LPS) did not significantly improve any parameter over the LPS group without PEEP in this ARDS model . LPS without PEEP (3.4 +/- 0.2 cells/6,400 micrometer2) caused a marked increase in the total number of sequestered leukocytes in the pulmonary parenchyma as compared with the control group (1.3 +/- 0.1 cells/6,400 micrometer2) . LPS + PEEP + Sf (2.3 +/- 0.2 cells/6,400 micrometer2) significantly decreased while LPS + PEEP significantly increased (4.0 +/- 0.2 cells/6,400 micrometer2) the total number of sequestered leukocytes as compared with the LPS without PEEP group . In summary, aerosolized surfactant replacement decreased leukocyte sequestration and improved oxygenation in our porcine model of endotoxin-induced lung injury.

Am J Physiol, 1998 Sep, 275(3 Pt 1), C740 - 7
Induction of Mn SOD in human monocytes without inflammatory cytokine production by a mutant endotoxin; Tian L et al.; Endotoxin selectively induces monocyte Mn superoxide dismutase (SOD) without affecting levels of Cu,Zn SOD, catalase, or glutathione peroxidase . However, little is known about the structure-activity relationship and the mechanism by which endotoxin induces Mn SOD . In this study we demonstrated that a mutant Escherichia coli endotoxin lacking myristoyl fatty acid at the 3' R-3-hydroxymyristate position of the lipid A moiety retained its full capacity to coagulate Limulus amoebocyte lysate compared with the wild-type E . coli endotoxin and markedly stimulated the activation of human monocyte nuclear factor-kappaB and the induction of Mn SOD mRNA and enzyme activity . However, in contrast to the wild-type endotoxin, it failed to induce significant production of tumor necrosis factor-alpha and macrophage inflammatory protein-1alpha by monocytes and did not induce the phosphorylation and nuclear translocation of mitogen-activated protein kinase . These results suggest that 1) lipid A myristoyl fatty acid, although it is important for the induction of inflammatory cytokine production by human monocytes, is not necessary for the induction of Mn SOD, 2) endotoxin-mediated induction of Mn SOD and inflammatory cytokines are regulated, at least in part, through different signal transduction pathways, and 3) failure of the mutant endotoxin to induce tumor necrosis factor-alpha production is, at least in part, due to its inability to activate mitogen-activated protein kinase.

Am J Respir Cell Mol Biol, 1998 Sep, 19(3), 470 - 6
Production of endothelins by the ventilatory muscles in septic shock; Guo Y et al.; Circulating endothelin-1 (ET-1) concentration increases significantly in animal models of sepsis . The main mechanism responsible for this rise in ET-1 levels is believed to be upregulation of ET-1 synthesis in various organs, such as the lungs and heart . In this study we investigated whether ET-1 is synthesized in the ventilatory muscles and whether this synthesis is regulated in septic shock . Conscious rats were injected with Escherichia coli endotoxin (lipopolysaccharide {LPS}) and killed 6, 12, and 24 h later . A fourth group of rats was injected with normal saline and served as a control . The diaphragm was excised at the end of the experiment and quickly frozen . Diaphragmatic ET-1 level was measured with radioimmunoassay, and messenger RNA (mRNA) expression of ET-1 precursor prohormone (preproET-1), preproET-3, and endothelin-converting enzyme was measured with reverse transcription-polymerase chain reaction . LPS injection elicited an early (within 6 h) and prolonged rise in diaphragmatic ET-1 concentration . In addition, mRNA levels of preproET-1 and preproET-3 rose by about 4- and 3-fold within 6 to 12 h of LPS injection, whereas mRNA of endothelin-converting enzyme increased by more than 10-fold and peaked within 24 h of LPS injection . Immunostaining with anti-ET-1 antibody revealed positive ET-1 staining in the endothelium and somatic muscle fibers of septic diaphragms . These results indicate that diaphragmatic muscle fibers synthesize significant amounts of ET-1 in septic shock and that the rise in ET-1 production is due to upregulation of ET precursors and the converting enzyme.

Biochemistry, 1998 Sep 8, 37(36), 12672 - 80
Functional comparison of two human monocyte chemotactic protein-2 isoforms, role of the amino-terminal pyroglutamic acid and processing by CD26/dipeptidyl peptidase IV; Van Coillie E et al.; Human Monocyte Chemotactic Protein (MCP)-2 has originally been isolated from stimulated osteosarcoma cells as a chemokine coproduced with MCP-1 and MCP-3 . Here, a 5'-end extended MCP-2 cDNA was cloned from a human testis cDNA library . It encoded a 76 residue MCP-2 protein, but differed from the reported bone marrow-derived MCP-2 cDNA sequence in codon 46, which coded for a Lys instead of a Gln . This MCP-2Lys46 variant, caused by a single nucleotide polymorphism (SNP), was biologically compared with MCP-2Gln46 . The coding regions were subcloned into the bacterial expression vector pHEN1, and after transformation of Escherichia coli, the two MCP-2 protein variants were recovered from the periplasm . The recombinant proteins were purified to homogeneity by heparin-Sepharose affinity chromatography and reversed-phase HPLC . Edman degradation revealed a Gln residue at the NH2 terminus instead of a pGlu . To evaluate the influence of the cyclization, this Gln was chemically converted into pGlu in both MCP-2 variants . The conversion was confirmed by electrospray mass spectrometry . rMCP-2Gln46 and rMCP-2Lys46 and the NH2-terminal cyclic counterparts were tested on monocytic cells in calcium mobilization and chemotaxis assays . No significant difference in biological activity was observed between the rMCP-2Gln46 and rMCP-2Lys46 isoforms . However, for both MCP-2 variants the NH2-terminal pyroglutamate was shown to be essential for chemotaxis, but not for calcium mobilization . NH2-terminal truncation of rMCP-2Lys46 by the serine protease CD26/dipeptidyl peptidase IV (CD26/DPP IV) resulted in the cleavage of the NH2-terminal Gln-Pro dipeptide, whereas synthetic MCP-2 with an amino-terminal pGlu remained unaffected . CD26/DPP IV-clipped rMCP-2Lys46(3-76) was almost completely inactive in both chemotaxis and signaling assays . These observations indicate that the NH2-terminal pGlu in MCP-2 is necessary for chemotactic activity but also that it protects the protein against degradation by CD26/DPP IV.

Biochemistry, 1998 Sep 8, 37(36), 12649 - 58
The mechanism of adenosylmethionine-dependent activation of methionine synthase: a rapid kinetic analysis of intermediates in reductive methylation of Cob(II)alamin enzyme; Jarrett JT et al.; Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine . During this primary turnover cycle, the enzyme alternates between the active methylcobalamin and cob(I)alamin forms of the enzyme . Formation of the cob(II)alamin prosthetic group by oxidation of cob(I)alamin or photolysis of methylcobalamin renders the enzyme inactive . Methionine synthase from E . coli catalyzes its own reactivation by a reductive methylation that involves electron transfer from reduced flavodoxin and methyl transfer from AdoMet . This process has been proposed to involve formation of a transient cob(I)alamin intermediate that is then trapped by methyl transfer from AdoMet . During aerobic growth of E . coli, electrons for this process are ultimately derived from NADPH, and electron transfer does not generate a detectable level of cob(I)alamin due to the large potential difference between the NADPH/NADP+ couple and the cob(I)alamin/cob(II)alamin couple . In this paper, we show that even in the presence of the strong reductant flavodoxin hydroquinone, cob(I)alamin is not observed as a significant intermediate . We demonstrate, however, that this is due to a rate-limiting reorganization of the cobalt ligand environment from five-coordinate to four-coordinate cob(II)alamin . Mutation of aspartate 757 to glutamate results in a cob(II)alamin enzyme that is approximately 70% four-coordinate, and reductive methylation of this enzyme using flavodoxin hydroquinone as the electron donor proceeds through a kinetically competent cob(I)alamin intermediate . Furthermore, wild-type cob(I)alamin enzyme produced by chemical reduction reacts with AdoMet in a kinetically competent reaction . We provide evidence that methyl transfer from AdoMet to cob(I)alamin enzyme results initially in formation of a five-coordinate methylcobalamin enzyme that slowly decays to the active six-coordinate methylcobalamin enzyme . We propose a kinetic scheme for reductive methylation of wild-type cob(II)alamin enzyme by adenosylmethionine and flavodoxin hydroquinone in which slow conformational changes mask the relatively fast electron and methyl transfer steps.

Biochemistry, 1998 Sep 8, 37(36), 12489 - 95
Investigation of the role of tyrosine-114 in the activity of human O6-alkylguanine-DNA alkyltranferase; Goodtzova K et al.; Tyrosine-114 is one of 13 totally conserved amino acids in all known sequences of O6-alkylguanine-DNA alkyltransferase (AGT) . The importance of this amino acid in repair of alkylated DNA by AGT was studied by changing it to phenylalanine (F), alanine (A), threonine (T), or glutamic acid (E) in human AGT . The activities of the mutant proteins were then compared to those of the wild type with regard to abilities to do the following: (a) protect Escherichia coli from the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); (b) repair methylated DNA in vitro; (c) bind to oligodeoxynucleotides containing O6-methylguanine; and (d) react with the low molecular weight pseudosubstrate, O6-benzylguanine . When expressed at high levels in E . coli strain GWR109, lacking endogenous AGT, the wild type and the Y114F mutant were highly effective in reducing mutations and cell killing by MNNG . The Y114A mutant had a much smaller protective effect, and mutants Y114T and Y114E were inactive . Purified preparations of all four AGT mutants showed an approximately similar degree (74-120-fold) of reduction in the rate of reaction with O6-benzylguanine . In contrast, the degree of reduction in activity toward methylated DNA substrates in vitro varied according to the mutation with the more conservative Y114F producing only a 30-fold reduction and the most drastic change of Y114E abolishing activity completely . Alteration Y114A produced a 1000-fold reduction whereas Y114T reduced activity by 10000-fold . All of the mutations affected the binding of AGT to single- or double-stranded oligodeoxynucleotides containing O6-methylguanine . The extent of increase in the Kd varied according to the amino acid with 2-5-fold (F), 7-11-fold (A), 167-200-fold (T), and 600-1000-fold (E) increases . These results are consistent with tyrosine-114 playing a role both in the binding of AGT to its DNA substrate and in facilitating the transfer of the alkyl group . It is probable that AGT resembles other DNA repair proteins in bringing about a "flipping out" of the target base from the DNA helix . Tyrosine-114 is therefore an excellent candidate for a key role in the interaction with the flipped O6-methylguanine . The results also show that when large amounts of AGT are produced in the cell, substantial decreases in the efficiency with which AGT can repair methylated DNA do not prevent the ability to protect E . coli from toxic alkylating agents . Mutant Y114F, whose activity was reduced by 30-fold, was equal to wild-type AGT in bringing about this protection.

Biochemistry, 1998 Sep 8, 37(36), 12458 - 64
Structural changes in the heme proximal pocket induced by nitric oxide binding to soluble guanylate cyclase; Zhao Y et al.; When expressed in Escherichia coli, the heme domain {beta1(1-385)} of rat lung soluble guanylate cyclase (sGC) is isolated with a stoichiometric amount of bound heme {Zhao, Y., and Marletta, M . A . (1997) Biochemistry 36, 15959-15964} . Nitric oxide (NO) binding to the heme in beta1(1-385) leads to cleavage of the Fe-His bond and formation of a five-coordinate NO-heme complex . Addition of imidazole to the five-coordinate NO complex shifts the Soret peak from 399 to 420 nm, which appears to result from the formation of a six-coordinate NO complex . Removal of the added imidazole by gel filtration results in formation of the five-coordinate NO complex once again . The EPR spectrum of the putative six-coordinate NO complex has nine distinct derivative-shaped lines (a triplet of triplets), which is the signature spectrum of a six-coordinate NO complex with two nitrogen atoms as the axial ligands . {15N}Imidazole simplifies the six-coordinate NO complex EPR spectrum to six distinct derivative-shaped lines (a triplet of doublets), indicating that the other axial ligand in the six-coordinate NO complex is an imidazole molecule . These results show that NO binding to sGC not only leads to the cleavage of the Fe-His bond but also induces a conformational change which opens the heme proximal pocket large enough to accommodate an exogenous imidazole molecule . These observations have important implications for determining the NO activation mechanism of sGC.

Biochemistry, 1998 Sep 8, 37(36), 12384 - 94
MutY DNA glycosylase: base release and intermediate complex formation; Zharkov DO et al.; MutY protein, a DNA glycosylase found in Escherichia coli, recognizes dA:dG, dA:8-oxodG, and dA:dC mismatches in duplex DNA, excising the adenine moiety . We have investigated the mechanism of action of MutY, addressing several points of disagreement raised by previous studies of this enzyme . MutY forms a covalent intermediate with its DNA substrate but does not catalyze strand cleavage . The covalent intermediate has a half-life of approximately 2.6 h, 2 orders of magnitude greater than the half-life of Schiff bases formed when E . coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III react with their respective substrates . The covalent complex between MutY and its DNA substrate involves Lys-142; however, the position of this residue in the presumptive active site differs from that of catalytic residues involved in Schiff base formation associated with endonuclease III and related DNA glycosylases/AP lyases . MutY converts DNA duplexes containing the dA:8-oxodG mispair to a product containing an abasic site; heat-induced cleavage of this product may account for the several reports in the literature that ascribe AP lyase activity to MutY . The MutY-DNA intermediate complex is highly stable and hinders access by Fpg to DNA, thereby avoiding a double-strand break . Cross-linking of MutY to DNA may play an important role in the regulation of base excision repair.

Neuroimmunomodulation, 1998 Sep-Oct, 5(5), 261 - 9
Clearance of {125I}-tumor necrosis factor-alpha from the brain into the blood after intracerebroventricular injection in rats; Chen G et al.; To test the hypothesis that brain to blood clearance is a mechanism by which brain inflammation and damage can increase circulating acute phase cytokines, rate of transfer of {125I}-tumor necrosis factor-alpha ({125I}-TNF) from brain to blood was determined . Acid precipitable {125I}-TNF appeared in peripheral blood within 5 min of intracerebroventricular (i.c.v.) injection and was cleared from brain to blood following first order kinetics at a fractional rate of 0.01123 +/- 0.0030/min, a value virtually identical with a previously determined clearance rate of {125I}-IL-6 . Area under blood concentration curve compared with that after intravenous injection shows that 31.6 +/- 5.8% of the intracerebral dose reached peripheral blood in 4 h . Elevated ratios of superior sagittal sinus to aortic blood radioactivity concentration at 1 and 3 h (1.48 +/- 0 . 26, p = 0.042; 1.95 +/- 0.39, p = 0.026, respectively) indicate that TNF-alpha drains from brain at least in part via the sagittal sinus . Escherichia coli lipopolysaccharide i.c.v . injection increased the rate of brain efflux of TNF-alpha.

Yeast, 1998 Aug, 14(11), 1017 - 25
AILV1 gene from the yeast Arxula adeninivorans LS3--a new selective transformation marker; Wartmann T et al.; The ILV1 gene of the yeast Arxula adeninivorans LS3 (AILV1) has been cloned from a genomic library, characterized and used as an auxotrophic selection marker for transformation of plasmids into this yeast . One copy of the gene is present in the Arxula genome, comprising 1653 bp and encoding 550 amino acids of the threonine deaminase . The protein sequence is similar (60.55%) to that of the threonine deaminase from Saccharomyces cerevisiae encoded by the gene ILV1 . The protein is enzymatically active during the whole period of cultivation, up to 70 h . Maximal activities, as well as protein concentrations of this enzyme, were achieved after cultivation times of 20-36 h . The AILV1 gene is a suitable auxotrophic selection marker in transformation experiments using an Arxula adeninivorans ilv1 mutant and a plasmid containing this gene, which is fused into the 25S rDNA of Arxula adeninivorans . One to three copies of the linearized plasmid were integrated into the 25S rDNA by homologous recombination . Transformants resulting from complementation of the ilv1 mutation can be easily and reproducibly selected and in addition are mitotically stable . Therefore, the described system is preferred to the conventional selection for hygromycin B resistance.

Scand J Infect Dis, 1998, 30(2), 169 - 72
Endotoxaemic liver injury in a porcine model--relation to tumour necrosis factor-alpha release and survival; Eriksson M et al.; The effects of a continuous infusion of E . coli endotoxin on liver blood chemistry, plasma TNFalpha and blood pressure were evaluated in relation to survival in 18 anaesthetized pigs . The endotoxin infusion cause both unconjugated and conjugated bilirubin to increase (both p < 0.001), as compared to 6 saline-infused controls . Four pigs died during the endotoxin infusion . The plasma levels of TNFalpha of these 4 non-survivors were higher compared to TNFalpha of the 14 surviving endotoxaemic pigs (p < 0.05) . The increase in plasma bilirubin of the non-survivors was less expressed (p < 0.05), and their arterial blood pressure and base excess deteriorated (both p < 0.001) compared to surviving endotoxaemic pigs . A decreased blood pressure explains both the reduced 'hepatic washout' of bilirubin and visceral ischaemia, reflected by the development of metabolic acidosis . This porcine model may be suitable for studying endotoxaemic liver injury.

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